|
http://www.abbs.info e-mail:abbs@sibs.ac.cn ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(2): 197-214 CN 31-1300/Q |
Abstracts
of the 5th A-IMBN ConferencePartial Abstracts of the 5th Conference of the
Asia-Pacific Infernational Molecular Biology Network (A-IMBN)(Part I)
Screening for
differentially expressed genes involved in the cytotoxicity of a 50 kD protein
purified from Tinospora rumphii Boerl on human cancer cell lines
( 1 Institute
of Biology, University of the Philippines, Diliman, Quezon City; 2 Research
and Biotechnology Division, St. Luke’s Medical Center, Quezon City,
Philippines; *e-mail: ffnatividad@stluke.com.ph )
The stem of Tinospora rumphii Boerl locally known as
makabuhay is one of the most common plants being used to treat various
ailments. In this study, a 50 kD protein was purified from the methanol extract
of the plant. The cytotoxic activity of the purified protein was evaluated by
the MTT assay. Different human cancer cell lines were treated with various
concentrations of the protein and the inhibitory concentrations (IC50)
were determined. The apoptosis-inducing activity of the protein was likewise
investigated. To characterize cell death, the same set of cell lines were
stained with acridine orange and ethidium bromide and viewed under fluorescence
microscope. Apoptotic index was determined and showed a dose- and
time-dependent curve. DNA fragmentation, a characteristic feature of apoptosis,
was also apparent.
Screening for differentially expressed genes is one
of the most straightforward approaches in understanding the molecular basis of
a biological activity. In this study, differential display reverse transcription
polymerase chain reaction (DDRT-PCR) was used to identify and characterize
genes that are expressed upon treating the human cancer cell lines with the 50
kD protein. Different populations of the extracted total RNAs were reversely
transcribed using anchored oligo-dT primers. PCR amplification of relative
cDNAs was carried out in combination with an arbitrary primer. The resulting
PCR products were subjected to electrophoresis using a denaturing 6% acylamide
sequencing gel. Results demonstrated a number of differentially expressed
transcripts in the treated groups. Sequence analysis showed that there were
more down-regulated genes than up-regulated genes.
Comparison of 2
strategies for isolating dengue virus in C6/36 A.albopictus cells by monitoring
growth patterns
C C Buerano1,2,
M Nepumoceno1, C Z Tanig2, J A Alfon2, D J
Cruz2, R R Matias1,2, F F Natividad2*
(1 University
of the Philippines, Diliman, Quezon City, Philippines; 2 St. Luke’s
Medical Center, Quezon City, Philippines; *e-mail: ffnatividad@stluke.com.ph )
Dengue virus is the causative agent of dengue
infection, manifestation of which ranges from a mild fever to the more severe
types, dengue hemorrhagic fever and dengue shock syndrome. Laboratory
confirmation of the disease is done by serology or detection and isolation of
the virus. Virus isolation is the best means of showing infection since most
serological tests are not reliably type specific. The most recommended method
for virus isolation is through inoculation to host cells such as such as the
more popularly used mosquito cell line C6/36 Aedes albopictus. Improvement in
the rate of virus isolation in this cell line include varying incubation period
which may range from 7 days to 14 days. In the present study, two strategies
for improving the rate of isolation were compared by monitoring the growth
patterns of the virus. In both strategies, serum samples known to contain
dengue virus were inoculated into culture tubes containing confluent C/36
cells. Infected cells were maintained in minimal essential medium containing 2%
fetal bovine serum. In the first strategy, the cells were incubated
continuously for 14 days. In the second strategy, cells were incubated for 7
days, after which spent fluid was removed, while the remaining cells were
re-cultured for another 7 days in fresh maintenance medium. To monitor growth
of the virus, aliquots of infected culture fluid were removed everyday until
the 14th day. ICF were then subjected to focus formation assay to detect
infectious virus particles. Viral growth patterns were graphed based on the
amount of infectious virus particles detected daily in the ICF. Results showed
that the re-culture of infected cells gave a higher success rate of virus
isolation with at a higher titer of focus-forming units.
( Virology
Lab, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China, *e-mail:
chengye1969@sohu.com )
Sugarcane or maize leaves with mosaic virus symptoms
were collected from 13 sites in China. Sequence data showed that all 8 samples
from maize contained Sugarcane mosaic virus (SCMV); complete sequences were
determined from 2 samples and partial sequences (the CI coding region and the
3’-part of the genome) from the others. All the 5 sugarcane samples contained a
virus tentatively described as Sorghum mosaic virus (SrMV), and in three of them,
SCMV was also detected; 2 SrMV sequences and 3 SCMV ones were completely
determined and partial SrMV sequences were obtained from the remaining 3
samples. The features of the complete sequences of SCMV and SrMV are described
for the first time. Sequence comparisons and phylogenetic analysis showed that
the Chinese SCMV sequences were or three distinct groups (sugarcane isolates
from Zhejiang province, a maize isolate from Guangdong and maize isolates from
other provinces). The SrMV sequences were similar to one another (>93%
identical nucleotides); they resembled published sequences in the coat protein
but were less similar in the 3’-UTR. The complete sequences of SCMV and SrMV
had about 70% nucleotides identical to one another and to Maize dwarf mosaic virus
(MDMV). MDMV was not detected in any of the samples.
Augmentation of
thermotolerance in primary skin fibroblasts from a transgenic pig
overexpressing the porcine HSP70.2
Ming-Yu Chen1,2*, Bor-Show
Tzang1, Ching-Fu Tu1, San-Yuang Huang1,
Jyh-Hung Lin1, Tzong-Hsiung Hseu2, Wen-Chuan Lee1
( 1 Division
of Biotechnology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan
35099, Miaoli, Taipei, China; 2 Department of Life Science,
Tsing-Hua University (Hsinchu), Taipei, China; *e-mail: cmy6075@seed.net.tw )
Heat shock protein 70 (HSP70) has been documented to
play an important role in cellular thermotolerance. We have recently reported
an association of mice thermotolerance with the induction of HSP70. However,
the direct evidence in whole animal for this phenomenon remains obscure. To
explore whether HSP70 is directly associate with thermotolerance, a recombinant
plasmid DNA containing the HSP70.2 and the reporter green fluorescence
protein gene was constructed under the control of cytomegalovirus enhancer and
promoter. The recombinant DNA was used to produce transgenic pigs
overexpressing porcine HSP70.2 by the technique of pronucleus
microinjection. The transgenic rate of F0 pigs and germ-line transmission rate
of F1 progeny were 16.7% and 57.9%, respectively. Expression of the transgene
in pigs was determined by PCR and immunoblot assay of ear tissue or lymphocytes
at the birth, 1 month, and 3 months of age. The primary fibroblast cell lines
were derived from four pigs, 2 transgenics and 2 non-transgenic littermates.
After heating at 45 ℃ for 3 h, the survival rate of the primary
fibroblast cells from a transgenic pig (78%) was proved to be higher (P=0.018)
than that of non-transgenic (63%). The results indicated a directed association
of HSP70 with the cellular thermotolerance from transgenic pigs.
( Karolinska
Institute Structural Virology, Department of Biosciences, 14157 Stockholm,
Sweden, *e-mail: holland.cheng@biosci.ki.se )
Dynamic biological processes are thought to be a
consequence of synergistic cooperativity and metastability. Critical
submolecular components of these complex machines often include local
environments of non-equivalence where dynamic pathways of action can only be
characterized by including less ordered transition states. Principles
describing these events were anticipated and articulated by far-sighted
structural biologists decades ago; however, it is only recently that
high-resolution structures of many of these large molecular machines could be
determined by cryoEM in combination with other means of structural analyses.
With rapid-freezing procedures, it is now possible to quantify chemical
descriptors of many biological processes in various degrees of detail. To
follow the conformation of subunit proteins in virus life cycles, cryoEM
provides snapshots of large-scale macromolecular assemblies The essences of
lateral interaction among protein subunits are studied by a lipid-enveloped
alpha virus, where glycoprotein interactions in the lipid membrane has a direct
role in guiding the infection process. In addition, the structure of
non-lipid-enveloped picornaviruses are also demonstrated regarding how they
utilize receptor to mediate capsid disassembly after re-entering the host cell
to subsequently release the genome.
( Institute of
Plant Protection, CAAS, Beijing 100094, China, *e-mail: zmcheng@95777.com )
The sequence of the GPV isolate of BYDV was
identified and its amino acid sequence was deduced. Genome organization has six
(+sense) open reading frames (ORFs) similar to those of RPV. In the
RNA-dependent RNA polymerase coding region, the length of ORF1 is 1953 nt and
encodes a 71.4 kD protein and ORF2 is 1872 nt and encodes a 70.1 kD protein.
There is a 601 nt overlap between ORF1 and ORF2. The coat protein of GPV
(molecular weight 22.2 kD) is encoded by ORF3 with a length of 603 nt. As MAV,
PAV and RPV, GPV contained a second ORF within the coat protein coding region.
This protein of 17.0 kD is thought to correspond to the VPg. The length of ORF4
is 453 nt long. ORF5, 1326 nt of length, is directly after the coat protein
termination codon, and in the same reading frame as the coat protein gene. This
protein is associated within the intact virus as a 72.2 kD protein.
The nucleotide and amino acid sequence homology of
GPV has a greater identity to the sequence of RPV than those of PAV and MAV.
The GPV ORF1 sequence shared 69% of nucleotide similarity and 57% of deduced
amino acid similarity with RPV-ORF1 whereas ORF2 shared 81% and 81%, ORF3 84%
and 77%, ORF4 87% and 86%, ORF5 69% and 69%, respectively.
An efficient pollen tube pathway-mediated transformation
protocol was developed for the generation of transgenic wheat plants that
express coat protein (CP) of BYDV GPV. Molecular analysis of the CP gene in
transgenic plants confirmed the stable integration of the CP gene into the
wheat genome and inheritance of the gene to T1, T2, T3 and T4 generations. Upon
inoculation with GPV in greenhouse tests, transgenic plants that expressed the
CP gene exhibited a significant delay in symptom development and reduced virus
accumulation compared with control plants by ELISA. In the field, our results
showed a higher level of resistance to virus infection in T2, T3 and T4 plants
indicating that the resistance trait was stably transmitted to transgenic T1,
T2, T3 and T4 plants.
Resistance evaluation of the T1 generation of
transgenic wheat plants carrying GPV replicase gene, were carried out in both
field and greenhouse conditions. Compared with non-transgenic plants, two
transgenic lines of Shan 160 showed high level of resistance with no or very
weak symptom on the leaf apex after virus infection. The transgenic line of
Longjian 127 also showed delayed symptom development, when the leaves of the
non-transgenic seedlings were all yellow, the flag leaves of the transgenic
line were still green, or with a yellowing area less than 1/3.
We have developed an improved protocol for the rapid
and efficient production of transgenic wheat with resistance to BYDV by using
no selectable markers and the pollen tube pathway to transfer the BYDV
replicase gene into the common wheat varieties, then sowing transgenic seeds
into the field for resistance screening and molecular assay. Transgenic wheat
plants resistant to BYDV GPV were obtained.
A comparison of
the epidermal cytokine pattern of patients with atopic dermatitits after
treatment with FK506 (Tacrolimus)
( Research and
Biotechnology Division; 1Immunology Center, St. Luke’s Medical Center, Quezon
City 1102, Philippines, *e-mail: ffnatividad@stluke.com.ph)
Atopic dermatitis is a chronic disease, which is
characterized by the proliferation of early and late inflammatory cells. This
process is influenced and favors the production of cells, cytokines and
adhesion molecules. FK506 is a recently discovered immunosuppressive drug,
which has a better safety profile than cyclosporin. Its mechanism of action is
similar to cyclosporin that it blocks T cell receptors and signaling,
decreasing T cell proliferation and IL2 secretion. The study population
consists of seven patients with moderate to severe atopic dermatitis. One
patient was treated with topical steroids and the six patients were placed on
FK506 treatment. Skin biopsy of all patients was performed prior to and after
application of FK506. mRNA was extracted from the samples and subjected to cDNA
synthesis and PCR using primers specific for IL4, IL5, IL13, gIFN, GM-CSF and
RANTES. mRNA expression level was measured using a hybridization capture assay
on a 96-well plate format. Preliminary results show a dramatic decrease in
inflammatory cytokine pattern in most patients treated with FK506. It was shown
that this drug was promising in the treatment of chronic inflammatory
dermatitis.
Mammalian
STE20-like kinase 2 (MST2) is regulated by a novel mechanism involving
proteolysis and phosphorylation
( Biochemistry
Department, Hong Kong University of Science & Technology; 1
Biochemistry Department, Hong Kong University of Science & Technology, Hong
Kong, China; *e-mail: jerwang@ust.hk )
Mammalian STE20-like kinase 2 (MST2) belongs to a
rapidly expanding protein kinase family, STE20-lik kinase family. Several
studies showed that MST2 or its close homolog MST1 was activated by
caspase3-catalyzed cleavage. A few studies showed that protein phosphorylation
also contributed to kinase activation. In this report, we examined the
mechanism of MST2 activation by both proteolysis and protein phosphorylation
reactions. Both the full length and caspase3-truncate from of MST2
over-expressed in cultured 293T cells were found to be active and in the
phosphorylated state. Incubation of the full length MST2 with protein
phosphatases resulted in rapid dephosphorylation of the protein with
concomitant decrease in kinase activity. Conversely, incubation of the cell
lysate with ATP-Mg2+ promoted further phosphorylation and activation of the
full length MST2. These observations suggest strongly that MST2 is activated by
protein phosphorylation. In contrast to full length MST2, the caspase3-truncate
form of the enzyme could not be significantly dephosphorylated, nor was it
further phosphorylated. The site of the activating phosphorylation is a unique
threonine residue, Thr180, at the kinase activation loop of MST2. Substituting
Thr180 in either the full length or the caspase3-truncate form of MST2 by
alanine rendered the kinase inactive. The phosphorylation of MST2 at Thr180
appears to be an autocatalytic reaction. The protein concentration dependence
of the auto-activation reaction is characteristic of an inter-molecular rather
than an intra-molecular reaction. Based on the results of the present study, a
novel mechanism may be proposed to account for the activation of MST2 during
apoptosis. Thus, MST2 kinase activity in cells is determined by the relative
rates of the auto-phosphorylation and the protein phosphatase reactions. At its
prevailing concentration in cells, MST2 is mostly in the dephosphorylated state
and therefore has low kinase activity. During apoptosis, caspase3-truncate form
of MST2 appears, which, due to its resistance to protein phosphatase reaction,
is largely in the phosphorylated state.
Molecular
cloning of CKLFSF1–8, 8 novel members of CKLF superfamily
Pei-Guo Ding, Wen-Ling Han*, Ming-Xu Xu, Lu Wang, Xiao-Yan Qiu, Min Rui, Ya-Nan Liu, Da-Long Ma
( Peking
University Center for Human Disease Genomics, Beijing 100083, China, *e-mail:
hanwl@bjmu.edu.cn )
CKLF is a novel gene located on chromosome 16, it consists 4 RNA splicing
forms, which are designated as CKLF1, 2, 3 and 4. CKLF2 is the
complete gene product and encodes 152 amino acids. By using bioinformatics, we
have successfully cloned rat and mouse CKLF2. The BLAST search in
combination with EST assembly using CKLF2 cDNA and protein sequences
identified 8 novel genes designated as chemokine-like factor superfamily member
1 – 8 (CKLFSF1 – 8). Computer-aided analyses and database searching reveal
that CKLF2 and CKLFSF1 – 8 have sequence identity between each
other. Most of them have alternative RNA splicing forms, and different isoforms
have different transmembrane regions. CKLF and CKLFSF1 – 4 exist
as a gene cluster on chromosome 16. CKLF, CKLFSF1, 2 are very
closely linked, the regions between CKLF, CKLFSF1 and CKLFSF1,
2 are less than 400 bp. The regulatory elements of CKLFSF1 transcription are
located on the CKLF gene, and CKLFSF1 has more than 16 isoforms
identified. CKLFSF6-8 form a gene cluster on chromosome 3 and CKLFSF5 is
mapped to chromosome 14. Based on the sequence identity, CKLF, CKLFSF1,
2 represent a subfamily, and CKLFSF3, 5 belong to another subfamily. For
their mouse homologues, CKLF and CKLFSF1 – 4 form a gene cluster
on chromosome 8, CKLFSF6 – 8 form a gene cluster on chromosome 9, CKLFSF5
is mapped to chromosome 14. For hCKLFSF2, there are two mouse homologues
of it on chromosome 8. Of all these members, CKLFSF4 is the most
conserved gene, all of the human, mouse and C.elegans CKLFSF4 have 208
amino acids, and the homology between human and mouse CKLFSF4 is up to
95% at the overall protein level, but CKLF and CKLFSF1,2 are
active during evolutionary progress. The CKLF gene products have some
characteristics associated with CC chemokine, but CKLFSF8 shares obvious
sequence similarity with plasmolipin, which was nominated as TM4SF11.
Primary functional analyses suggest they may play important roles in the
process of cell proliferation and differentiation in immune system,
reproductive system and CNS. It appears that CKLF2 and CKLFSF1 –
8 represent a novel gene family links chemokines and transmembrane proteins.
Further studies of them should give us more interesting results.
This work was supported by the National High
Technology Research and Development Program of China (863 Program) (No.
2001AA215061).
Induction of
apoptosis by N-phosphoryl dipeptide methyl esters in K562 cells
( Key Laboratory of Bioorganic Phosphorus Chemistry, Ministry of Education, Department of Chemistry, School of Life Sciences and Engineering, Tsinghua University, Beijing 100084, China, *e-mail: duwei00@mails.tsinghua.edu.cn )
As a mechanism of deleting cells from tissues,
Apoptosis plays an important role in both physiological and varieties of
pathological situations, especially cancer conditions. In order to search for
tumor cells apoptosis inducers, a series of N-phosphoryl dipeptide methyl
esters were synthesized and their inhibition effects on K562 cell lines were
studied by MTT assays. The result showed that (DIPP-Trp)2-Lys-OCH3，DIPP-Trp-Met-OCH3
and DIPP-Met-Trp-OCH3 were the compounds that had better activity
and their IC50 were 39.0 mg/L, 64.5 mg/L and 75.2 mg/L. Under optic
microscope and fluorescence microscopy, the typical morphological changes of
apoptosis of K562 cells were observed when incubated with these N-phosphoryl
dipeptide methyl esters, strongly hinted that they might exert their anticancer
activity by inducing K562 cells apoptosis.
Detection of
overexpressed MDR1 mRNA using reverse transcriptase-polymerase chain reaction
L M Florento1,3*, R R Matias1,2
( 1
United Laboratories, Inc. Mandaluyong City, Philippines; 2
Research & Biotechnology Division, St. Luke’s Medical Center, Philippines; 3
Graduate School, University of Santo Tomas, Philippines; *e-mail: leilaflorento@yahoo.com
)
The MDR1 gene responsible for multi drug
resistance in human cells encodes a broad specificity efflux pump known as
P-glycoprotein. Overexpression of the MDR1 mRNA is reported to be an
important determinant of the response to chemotherapy and survival in some
cancers. The objective of the study is to detect overexpressed MDR1 mRNA
using RT-PCR. The complete genome sequence of the MDR1 mRNA was
downloaded from the NCBI (GenBank) database. Based on the consensus sequence,
two pairs of oligonucleotide primers were designed using the DNasis and Oligo
6.0 software to amplify a 1078 bp fragment of the MDR1 mRNA. Amplified
products were visualized by agarose gel electrophoresis followed by ethidium
bromide staining. Preliminary studies were done on HT-29 and IM-9 cell lines.
Since the clinical significant level of expression has to be defined, an
internal control for the quantitative RT-PCR is currently being developed.
Jian-Lin Fu*, Chay Boon
Loh, Kuo Ming Lip, Wai Hong Yuen, Shyh Shiuann Wu, Qiu-Rong Xu
( Institute of
Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, *e-mail:
mcbfujl@imcb.nus.edu.sg )
Amphibious frogs (Xenopus) are vertebrates used as animal model. They are easily maintained in the laboratory and can be induced by simple hormone injection to produce large number of eggs. The large size of their eggs allows micromanipulation and mircoinjection easily. The embryos are optically transparent and rapidly develop to facilitate investigations. These features make frogs Xenopus excellent animals for analyzing early development. During the past several years, many new techniques have been devised or adapted for Xenopus. Transgenesis in Xenopus embryos has proven useful for various studies. These include regulation of gene promoters from many organisms, labelling specific structures in vivo, study of misexpress genes during development with better spatial and temporal control, study of the molecular basis of later development events such as organogenesis, and generation of mutations in genes using gene trap approaches. Current interest is to study the regulation of gene promoters, using the green fluorescent protein (GFP) as a marker, and cooperate with the groups in our institute. The promoters experimentally used are from Fugu, Mouse, Rat, Human Drosophilia and Snail. These promoters were analyzed in live Xenopus laevis embryos. The transgenic embryos can be generated one day and analyzed the next. Some promoters showed the tissue specific characters. Green fluorescence was visualized in eye, muscle, brain, neuron, and ubiquitous separately, by UV microscope and photographed by digital camera. Transgenic frog system is still in its infancy in developmental biology.
Frank Gannon*, George Reid,
Michael Huebner, Raphael Metivier
( Executive
Director, EMBO, Meyerhofstrasse 1, D-69117 Heidelberg, Germany, *e-mail:
Frank.Gannon@embo.org )
The Estrogen Receptor is a key molecule in normal
physiology and in a variety of pathological conditions including breast cancer.
Studies carried out to date have focussed on specific interactions of the
Estrogen Receptor with the target genes that it activates. We have undertaken a
complex analysis on the behaviour of the Estrogen Receptor in cells under
variety of different conditions. We have shown that the Estrogen Receptor is
degraded by the proteasome following ubiquitination. Inhibition of degradation
by the proteasome unexpectedly blocks transcription of the genes that are
normally induced by the Estrogen Receptor. Conversely the inhibition of
transcription blocks the degradation of the Estrogen Receptor by the
proteasome. These data were extended by the use of FRAP (fluorescence recovery
after photobleaching) and chromatin immunoprecipitation (ChIP) revealing that
the Estrogen Receptor cycles on to and off target genes in both the presence
and the absence of its ligand estradiol.
These data provide a new integrated view of the
actions of estradiol in target cells that can have a bearing on the way in
which cancers in these cells should be treated in the future.
The icosahedral
glycoprotein shell of Semliki Forest virus controls both virus budding and
membrane fusion
Henrik Garoff*, L Haag, K
Forsell, L Xing, T Kozlovska, L Hammar, S T Kan, R H Cheng
( Department
of Biosciences, Karolinska Institute, Stockholm, Sweden, *e-mail:
henrik.garoff@cbt.ki.se )
In the icosahedral (T=4) Semliki Forest virus, the
envelope protomers, that is E1-E2 heterodimers, make one-to-one interactions
with capsid proteins below the viral lipid bilayer, transverse the membrane and
form an external glycoprotein shell with projections. The shell is organized by
protomer domains interacting as hexamers and pentamers around shell openings at
icosahedral 2- and 5-fold axes, respectively, and the projections by other domains
associating as trimers at 3- and quasi 3-fold axes. The virus is formed by
budding at the plasma membrane and enters into new cells by fusing its membrane
with that of the endosomal membrane. We have studied the role of the
glycoprotein shell in the budding and the fusion reactions using biochemical,
genetic and cryo-electron microscopy techniques. Studies with a virus mutant
that was defective in forming its nucleocapsid demonstrated that the
glycoproteins contained the information for the formation of an icosahedral
virus although they had to be triggered by an interaction with the capsid
proteins. Furthermore, we have found that low pH, as it occurs in the endosomes
during virus uptake, results in the relaxation of protomer interactions around
the 2- and the 5-fold axes in the shell and movement of protomers towards 3-
and quasi 3-fold axes in a way that reciprocally relocates their putative E1
and E2 domains. This seemed to be facilitated by a trimerization of
transmembrane segments at same axes. The alterations observed help to explain
several key features of the spike mediated membrane fusion reaction, including
shell dissolution, heterodimer dissociation, fusion peptide exposure and E1
homotrimerization. Altogether the results suggest that the glycoprotein shell
controls both the virus budding and the membrane fusion reactions.
Fumio Hanaoka1,2,3*
( 1
Grad. Sch. Front. Biosci., Osaka Univ., 1-3 Yamada-oka, Suita, Osaka 565-0871,
Japan;
2 Core Res. Evol. Sci. Tech., Japan Sci.
Tech. Corp., 4-1-8 Honmachi, Kawaguchi, Saitama 332-0012, Japan;
3 Cell Physiol. Lab., RIKEN, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan;
*e-mail: fhanaoka@fbs.osaka-u.ac.jp )
During the past several years, a new superfamily of
DNA polymerases called the Y-family has been identified. This group includes
evolutionarily related proteins known to be involved in mutagenesis. Several of
these enzymes support replicative bypass of damaged bases that arrest high
fidelity, highly processive DNA polymerases involved in DNA replication.
This process (translesion DNA synthesis) lies at the
center of mutagenesis. Human DNA polymerase η (Pol η)
was found to be a gene product responsible for an autosomal recessive disease,
namely the variant form of xeroderma pigmentosum (XP-V), characterized by a
high incidence of sunlight induced skin cancer. Pol η
belongs to the Y-family of DNA polymerases.
At first sight it may appear paradoxical that the
inactivation of a low fidelity DNA polymerase, such as Pol η
in XP-V cells, renders cells hypermutable to UV light. This paradox is best
understood on the basis of the capacity of Pol η
to read efficiently through the major UV lesion, a TT cyclobutane dimer, by
inserting two adenines across the dimer thus restoring the correct sequence.
Mechanisms of apparent accurate translesion DNA
synthesis by human Pol η as well as possible biological role(s) of
the Y-family DNA polymerases will be discussed.
Structural and
functional study of a novel cytokine CKLF1 and it variant CKLF2
Wen-Ling Han*, Dong-Lan Xia, Ya-Xin Lou, Min Rui,
Ying-Mei Zhang, Ya-Xia Tan, Da-Long Ma
( Peking
University Center for Human Disease Genomics, Beijing 100083, China, *e-mail:
hanwl@bjmu.edu.cn )
CKLF1(Chemokine-like factor 1) is a novel cytokine
isolated from PHA-stimulated U937 cells by SSH method whose expression level
could be partially inhibited by IL-10. CKLF1 has 3 other RNA splicing forms
that were named CKLF2, 3 and 4; CKLF2 is the full cDNA product of CKLF gene.
CKLF1 and CKLF2 have higher expression level in many tissues. The CKLF
gene is mapped to chromosome 16; it contains 4 exons and 3 introns. CKLF1
has a CC characteristic structure and shows identical key residues near the CC
motif with TARC and STCP-1, the only two CC chemokines located on chromosome
16. Signal P analysis indicates that CKLF1 has no typical signal peptide.
Subcellular localization and western blot analyses verified that CKLF1 could be
secreted into the supernatants of cell culture. Human CKLF1 has chemotactic
effects on lymphocytes, neutrophils and monocytes both in vitro and in vivo;
it can stimulate the regeneration of skeletal muscle cells and cause
inflammation changes of lung in vivo. To probe the function of CKLF
during myogenesis, C2C12 myoblast was stably transfected with the hCKLF2
eukaryotic expression vector. Compared with control vector transfected C2C12
myoblast, hCKLF2 over expression caused accelerated myoblast proliferation and
differentiation, which was determined by higher expression level of myogenin,
creatine kinase, myosin and myoblast fusion Overexpression of hCKLF2 resulted
in distinct myotube formation even in the high-serum medium. These findings
strongly suggest a direct role for hCKLF in regulation of skeletal muscle cell
growth. In addition, hCKLF2 can promote the proliferation and exert an
anti-apoptotic effect on BALB/C 3T3 cells. Based on the cDNA and protein
sequences of hCKLFs, we successfully cloned rat CKLF1, 2 and mouse CKLF2, 4
which have similar structure and functional characteristics with human CKLFs.
Therefore, we have found a unique cytokine with chemotactic effects on
leukocytes as well as important functions on cell proliferation,
differentiation and apoptosis.
This work was supported by the National High
Technology Research and Development Program of China (863 Program)
(No.2001AA215061).
p53, mitochondria and drug development
T Hara, N.
Tsuchida１, Katsuro
Koike*
( The Cancer
Institute, JFCR, Kami-Ikebukuro Toshima-ku, Tokyo 170-8455, Japan; １Tokyo Medical and Dental University, Yusima Bunkyo-ku, Tokyo 113-8549,
Japan; *e-mail: kkoike@jfcr.or.jp )
Our previous reports have indicated that
mitochondrial dysfunction and aggregation are associated with cell death in
response to the HBV X gene expression, by which a transcription factor p53 is
translocated from the nucleus to the aggregated mitochondria. Lactacystin is
known to be a proteasome inhibitor that can inhibit the p53 degradation and
induce mitochondrial aggregation, resulting in cell death. We examined the
intracellular location of p53 in Lactacystin-treated cells. Wild-type p53
became associated with aggregated mitochondria in p53 gene-transfected
Saos-2 cells in the presence of Lactacystin. Endogenous mutated p53 in Huh-7
cells was also shown to behave similarly like p53 in the p53
gene-transfected Saos-2 cells. Moreover, we observed that cells possessing
endogenous mutated p53 were more susceptible to proteasome inhibitor-dependent
apoptosis than p53 deficient cells. Then, we carried out in vitro cyt.c release
assay in order to examine whether p53 on the mitochondria directly triggers
cell death. It was clearly observed that cyt.c was released from the
mitochondria in a dose-dependent manner. Collectively, the data indicate that
mitochondrial p53 is directly involved in the apoptotic process, where p53
translocation to the aggregated mitochondria may be implicated in the mechanism
to trigger cell death under the proteasome-inhibited conditions.
Taken together with our data, we will discuss why and
how p53 acts on the mitochondria under the conditions of proteasome inhibitor-dependent
apoptosis. Since our data suggest that the mitochondria is a therapeutic target
for mutated p53-expressing tumors, we will also discuss a possibility that a
method of treatment that attacks the p53-mutated cancerous cells using
proteasome inhibitors as an antitumor agent can be established.
PCR-detection
of CEA mRNA in regional lymph nodes ofpatients with colorectal cancer
Mignonette S
Hipolito1, Edgardo M Bondoc3, Ronald R Matias1,2,
Pia Donna N Lorena2, Colorectal Cancer Study Group2, Filipinas F Natividad１*
( 1
Institute of Biology, University of the Philippines, Diliman, Quezon City,
Philippines; 2 Research and Biotechnology Division; 3
Institute of Digestive Diseases, St. Luke’s Medical Center, E.
Rodriguez Blvd., Quezon City, Philippines; *e-mail: ffnatividad@stluke.com.ph )
The prognostic potential of RT-PCR on
carcinoembryonic antigen (CEA) determination was investigated and compared with
the histopathological method for lymph node status determination in colorectal
cancer.Detection of CEA by RT-PCR was performed on lymph nodes taken from
patients with colorectal cancer and benign colorectal diseases. Lymph nodes
were resected from pericolic and peritumoral regions and total RNA was extracted
from each node separately. Primers specific for the CEA gene were used and the
presence of CEA messenger ribonucleic acid (mRNA) in lymph node samples
was considered evidence of metastasis.
35 or 37% of 94 mRNA samples obtained from
histologically negative lymph nodes were found positive for CEA by RT-PCR.
Lymph nodes from a patient with benign colorectal disease exhibited no CEA
mRNA. Overall, 43 (44%) of the total 102 lymph nodes were positive by RT-PCR
detection compared with only 8 (7.8%) lymph nodes positive for micrometastases
by histopathological analysis. This study shows that RT-PCR of CEA mRNA in
patients with colorectal cancer may provide additive value to histopathological
analysis in detecting lymph node micrometastasis and predicting recurrence,
especially when micrometastasis spreads out extensively from the main tumor to
distant lymph nodes.
Analyses of
neural stem cell lines established from fetal brains of p53-/-
mice
Makoto
Horiuchi, Yasuhiro Tomooka*
( Department
of Biological Science and Technology, Tokyo University of Science, 2641
Yamazaki, Noda, Chiba 278-8510, Japan, *e-mail: tomoylab@rs.noda.tus.ac.jp )
The developmental process of the central nervous
system (CNS) is not well understood. Especially the mechanism is poorly
explained how neural stem cells generate neurons, astrocytes and
oligodendrocytes. Neural stem cell lines (NSC) are useful tools for
investigating molecular mechanisms that regulate the neural cell diversity. In
the present conference, we introduce two NSC lines designated FBD-103a and
FBD-104. They are clonal lines established from fetal brains of p53-/-
mice. Western and Northern blot analyses revealed that FBD-103a cells express
nestin, a marker of undifferentiated neural cells. They also express cell-type
specific markers of neurons (neurofilament, NF), astrocytes (glial fibriallary
acidic protein, GFAP) and oligodendrocyte (myelin basic protein, MBP),
suggesting that the line contains multipotent stem cells. FBD-103a cells were
recloned and 19 subclones were established. Ten subclones express all of NF,
GFAP and MBP. Three subclones express both GFAP and MBP. Five subclones express
only NF, and one subclone expresses none of them. The results indicate that the
original FBD-103a cell was a multipotential stem cell with capability of
producing neuron-restricted precursors and glial-restricted precursors.
FBD-104 cells express markers of both mature and
immature astrocytes, such as GFAP, vimentin and nestin. They do not express
markers for neurons and oligodendrocytes when cultured in serum-containing
medium. However, they form spheres and express Mash-1, a bHLH-type neurogenic
transcription factor, when grown in suspension culture in serum-free medium
containing bFGF and EGF. These results suggest that FBD-104 cells are like NSCs
with potential for neurogenesis and some properties of astrocytes as NSCs shown
in the subventricular zone of the adult brain neocortex. These two lines will
provide useful models to gain further insight into molecular mechanisms in
neural cell diversity in mammalian CNS.
Characterization
of shrimp white spot syndrome viral proteins using proteomic approaches
( Department
of Biological Sciences and Tropical Marine Science Institute, National
University of Singapore, Singapore 119260, *e-mail: dbshead@nus.edu.sg )
White spot syndrome virus (WSSV) is at present one of
the major pathogens in shrimp culture worldwide. The complete genome of this
virus has been sequenced recently. To identify the structural and functional
proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four
protein bands were excised, in-gel digested with trypsin, and subjected to
matrix-assisted laser desorption ionization-time of flight mass spectrometry
and electro-spray ionization tandem mass spectrometry, respectively. Eighteen
proteins matching the open reading frames of WSSV genome were identified.
Except for three known structural proteins and collagen, the functions of the
remaining 14 proteins were unknown. Temporal analysis revealed that all the
genes were transcribed in the late stage of WSSV infection except for vp121. Of
the newly identified proteins, VP466 (derived from band 16) was further
characterized. The cDNA encoding VP466 was expressed in E.coli as a glutathione
S-transferase (GST) fusion protein. Specific antibody was generated with the
purified GST-VP466 fusion protein. Western blot showed that the mouse
anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV.
Immuno-gold labeling revealed that VP466 protein is a component of the viral
envelope. Results in this investigation thus proved the effectiveness of
proteomic approaches for discovering new proteins of WSSV.
Expressional and functional analyses of several gene
families isolated from developing cotton fiber
Sheng-Jian Ji,
Yong-Hui Shi, Yu Xu, Yu-Xian Zhu*
( State Key
Laboratory of Protein Engineering and Plant Genetic Engineering, College of
Life Sciences, Peking University, Beijing 100871, China, *e-mail:
zhuyx@water.pku.edu.cn )
With 10 DPA(days post anthesis) upland cotton fiber and 10 DPA ovule of a fuzzless-lintless mutant of upland cotton (fl), we isolated a group of cDNAs differentially expressed in cotton fiber using PCR-Select cDNA Subtraction method. From these cDNAs, several important gene families were identified: proline-rich protein, arabinogalactan protein, expansin, a-tubulin, b-tubulin, lipid transfer protein, and b-ketoacyl CoA synthase. Differential screening based on cDNA macroarray technology and semi-quantitive RT-PCR analysis verified that they were expressed only in developing fiber or much higher than in fl mutant ovule. Their expression patterns during early fiber development were studied. The high accumulation of their transcripts in elongating fiber implied that they might play roles in fiber development, especially in fiber elongation. In situ hybridization analysis of a b-tubulin Gh-BTubL revealed that there was no Gh-BTubL mRNA in fl ovule while it was easily detected in the elongating wild type cotton fiber cells. Overexpression of Gh-BTubL in fission yeast induced longitudinal growth of the host cells by 1.74-fold with no apparent effect on other aspects of the host cells. We suggest that Gh-BTubL plays an important role in cotton fiber elongation and we believe that full elucidation of these gene families may help improve our understanding of fiber development.
Chirality in the condensed phase: Development of a
novel instrument and its application to crystal chirality of inorganic salts
and films of proteins
( The
University of Tokyo, Department of Life Sciences, Graduate School of Arts and
Sciences, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan; and Kuroda
Chiromorphology Project, ERATO, JST, Park bldg., 4-7-6 Komaba, Meguro-ku, Tokyo
153-0041, Japan; *e-mail: ckuroda@mail.ecc.u-tokyo.ac.jp )
Solid-state spectroscopy provides valuable
information on solid-state structure and supuramolecular properties that are
not obtainable from the solution phase. However, very few reliable chirality
measurements in the solid state have been reported to date. This is because CD
spectra in the solid state suffer from artifacts which originate from the
macroscopic anisotropies of a sample such as LB (Linear Birefringence) and LD
(Linear Dichroism) and their interaction with the non-ideal characteristics of
polarization-modulation instruments, and in the case of powdered crystals,
dispersion effect. To obtain true CD spectra, we have designed and constructed
a Universal Chiroptical Spectrophotometer (UCS) which can deal with macroscopic
anisotropies of solid samples. We have also devised several procedures to
cancel out the artefacts, Using the novel instrument, we have measured
chirality of α-Ni(H2O)6·SO4
which exhibits optical activity only in the crystalline state due to chiral
supramolecular arrangement of non-chiral components, and other zinc porphyrin
complexes.
The work has extended to CD spectroscopy of proteins
in dry thin films to study their unique conformation or an aggregate form. The
information may be particularly relevant to some neurodegenerative disorders
such as Alzheimer and prion diseases where abnormal aggregates of β-amyloid
peptide or prion protein seem to constitute an important step. Solid-state CD
of bovine serum albumin in dry thin films was reported to exhibit different
conformation in the film, exhibiting different spectra from that in solution.
Using our instrument, we could show the sample exhibit different spectra
depending on the sample preparation method and the rotation positioning of the
film. It was revealed that this is due to the macroscopic anisotropies of the
samples and not due to the protein conformation transition during the film
formation, as suggested in the literature.
Enrichment
enhances the expression of sgk, a glucocorticoid-induced gene, and reverses sgk
mutant DNA-induced memory deficits
( Institute of
Biomedical Sciences, Taipei, China, *e-mail: eminy@ibms.sinica.edu.tw )
By using the PCR differential display method, we have
recently identified 98 cDNA fragments that are differentially expressed between
the fast learners and slow learners in the Morris water-maze learning task. One
of these cDNA fragments encodes the rat serum- and glucocorticoid-inducile kinase
(sgk) gene. Both northern blot analysis and in situ hybridization
histochemistry revealed that the sgk mRNA level is significantly higher
in the hippocampus of fast learners than slow learners. Transient transfection
of the sgk mutant DNA to the hippocampus impaired, whereas transfection
of the sgk wild-type DNA facilitated water maze performance in rats.
These results suggest that sgk plays a causal role in learning and memory
processes in rats. On the other hand, several studies have revealed that
animals reared in an enriched environment show improved learning and memory
performance in a variety of behavioral tasks. These results suggest that sgk
may play an important role underlying enrichment-induced memory facilitation.
The present study examined this hypothesis. Our results revealed that rats
reared in an enriched environment showed a significant increase in sgk mRNA
level in the hippocampus by real-time PCR analysis. This result is confirmed by
western blotting that SGK protein level is markedly increased in enriched rats.
Further, the effect of environmental enrichment on sgk expression is
specific to the hippocampus and not observed in other brain areas. We then
studied the functional significance of enrichment-induced sgk expression.
Upon transfection of the sgk mutant DNA to the hippocampus, we found
that the performances of spatial-learning, fear-conditioning memory and
object-recognition memory were significantly impaired in rats. However, these
spatial and non-spatial memory deficits were all reversed upon environmental
enrichment training. These results together suggest that sgk may serve as a
molecular mechanism of enrichment-induced learning and memory facilitation.
( Department
of Biochemistry, Hong Kong University of Science and Technology, Clear Water
Bay, Kowloon, Hong Kong, China, *e-mail: bccliang@ust.hk )
Eukaryotic cells duplicate their genome in the S
phase of the cell cycle by initiating DNA replication at multiple chromosomal
sites called origins of DNA replication. Initiation of DNA replication is
controlled by the cis-acting DNA elements called replicators and the
trans-acting initiation proteins that interact with the replicators.
Among all eukaryotes, DNA replication has been most extensively studied in the budding yeast Saccharomyces cerevisiae. The replicators and many initiation proteins and their regulators have been identified, and a framework of interactions among these factors has been elucidated in S. cerevisiae. On the other hand, the mechanisms of action of many initiation proteins remain unknown. Moreover, some initiation proteins have not been discovered.
We have been performing genetic screens to identify
previously unknown initiation proteins for yeast DNA replication. We have
identified several proteins that interact with ORC and MCM proteins by
screening for multicopy suppressors of orc and mcm mutants. A novel initiation
protein we identified is Noc3p(nucleolar complex associated protein). Noc3p
interacts with MCM proteins and ORC and binds to chromatin and replicators
throughout the cell cycle. It functions as a critical link between ORC and
other initiation proteins to effect chromatin-association of Cdc6p and MCM
proteins for the establishment and maintenance of pre-replication complexes. We
have also developed a comprehensive phenotypic screen by novel adaptation of
several genetic strategies, including colony color assay and plasmid loss assay
with a pair of plasmids, one of which contains a single ARS while the other
contains multiple ARSs.
To develop potential anticancer agents, we have
designed, screened and identified antisense oligonucleotides that can inhibit
the expression of replication initiation proteins and induce apoptosis in human
cancer cells.
This work was supported by the Hong Kong Research
Grants Council.
Immunoglobulin
A and immunoglobulin G Expression in mouse colostrum-forming mammary gland
epithelial cells
Yun-Tao Mao, Xiao-Yan Qiu*, Xiao-Hui Zhu, Le-Meng Wu, Xin Sun,
Da-Long Ma
( Peking
University Center for Human Disease Genomics, 38 Xue-Yuan Road, Beijing 100083,
China, *e-mail: qiuxy@bjmu.edu.cn )
Milk contains a multitude of components primarily of
the sIgA and IgG that can provide immune protection to the suckling offspring.
In addition, these specialized factors are essential for the protection of the
mammary gland from pathogen colonization and lactation failure. To the present
studies, this process is primary dependent on a highly selective mechanism by
which the circulating precursors of mucosal IgA plasma cells selectively lodge
in mammary gland. However, the number of stromal plasmacytes and lymphocytes in
lactating mammary gland is small, which made the above mechanism highly
doubtable. Enlightened by Qiu’s discovery of immunoglobulin expression and its
gene rearrangement in malignant epithelial tumor cells, to detect whether the
mammary gland epithelial cells expressed Ig themselves, H&E staining,
immunohistochemistry staining, RT-PCR, Northern blotting analysis, and in situ
hybridization were performed. The results were as following: (1)H&E
staining presented the number of lymphocytes and plasmacytes in colostrum-forming
mammary gland is small indeed; (2)By using biotinylated goat anti-mouse IgA and
IgG as primary antibodies, immunohistochemistry staining localized IgA and IgG
in the milk in lactiferous ducts and most mammary gland acinar epithelial
cells; (3)Expressions of IgA and IgG in mammary gland were detected using
RT-PCR, amplification fragments were analyzed by DNA sequencing, and the
abundance of IgA was higher than housekeeping gene G3PDH;
(4)Transcript levels of IgA and IgG were investigated by Northern blotting and
strongly positive signals were detected in mammary gland using IgA and IgG 32P-labeled
cDNA probes; (5)Specific IgA and IgG transcripts detection in mammary gland
epithelial cells was done with DIG-labeled cRNA probes by in situ
hybridization.
Ronald R Matias1,2*, Filipinas F
Natividad2, Ma Isolde Caperig2, Clyde P Dapat2,
Dengue Study Group2
( 1 Institute
of Biology, College of Science, University of the Philippines; 2
Research and Biotechnology Division, St. Luke’s Medical Center, Philippines; *e-mail:
rrmatias@stluke.com.ph, rrm@nib.upd.edu.ph )
Epitopes of dengue virus were identified from a random
peptide phage library using patients’ sera with high titers of Immunoglobulin M
(IgM). Three rounds of panning were performed in order to select and amplify
phage clones displaying 12-mer random peptides that bind to immobilized IgM. 35
phage clones were totally isolated by ELISA. Phage DNA of these immunopositive
clones was prepared and its sequence was determined. The deduced amino acid
sequence showed that there were 13 clones having unique sequences. Three of
these clones, 313C-E7, -G4, and -F5 have a consensus sequence of PKaPbEx (a=P/T
and b=A / S). The peptide sequence of clone 313C-G4 is similar to the envelope
protein of dengue serotypes 1, 2 and 4 at positions 649 – 658. Among the 13
clones, four peptides were synthesized and characterized by indirect
competition ELISA.
( Institute of
Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan; Kanagawa Academy of Science and Technology,
907 Nogawa, Miyamae-ku, Kawasaki 216-0001, Japan; *e-mail:
miyajima@ims.u-tokyo.ac.jp
)
Liver is a center for metabolism in adult body.
However, fetal liver lacks most of such activity and functions as the major
hematopoietic tissue until birth. In order to study fetal liver hematopoiesis
as well as liver development, we have developed a culture system of mouse
embryonic day 14.5 liver cells. In this system, differentiation of fetal
hepatocytes can be induced by Oncostatin M (OSM), a member of the IL-6 family
cytokines, as evidenced by morphological changes and expression of various
adult liver enzymes as well as functions such as ammonia clearance, glycogen
accumulation and lipid synthesis. This culture system also supports
proliferation and differentiation of hematopoietic stem cells, recapitulating
fetal liver hematopoiesis. Interestingly, OSM is expressed in hematopoietic
cells in fetal liver, suggesting that hematopoietic cells supported by fetal
hepatic cells in turn induces differentiation of hepatocytes by producing OSM.
Thus, OSM appears to play a role for coordinating development of liver and
hematopoiesis. By using this system, we have been studying the mechanism of
liver development and found that OSM-induced STAT3 activation plays a major
role for induction of various hepatic enzymes, while K-Ras mediates an
OSM-induced signal for the formation of adherens junction. Studies on the
mechanism of liver development will be also discussed.
Possible
applications of conditionally immortalized tissue cell lines with
differentiation functions
( Department
of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University,
4-1, Seiryomachi, Aoba-ku, Sendai, Miyagi 890-8575, Japan, *e-mail:
mobinata@idac.tohoku.ac.jp
)
If all distinct cell types of the body could be
clonally isolated and stocked, similar to cDNA or genomic DNA libraries, it
would be very invaluable for studying the tissue and cellular functions. We
developed a new method to establish conditionally immortalized cell lines that
retain the differentiated cell functions similar to the original tissues, using
the temperature-sensitive(ts) simian virus 40 large tumor antigen (SV40 large
T-antigen) gene transgenic mouse. We successfully established many functional
cell lines including hepatocyte, renal tubule cell, vascular smooth muscle
cell, gastric surface mucous cell and various bone marrow stromal cells. We
developed in vitro hematopoietic microenvironment to support growth and
differentiation of hematopoietic stem cells and their progenitors by co-culture
with bone marrow stromal cells. In addition, we showed that bone marrow stromal
cell lines have properties of mesenchymal stem cells that differentiate towards
osteoblast, skeletal muscle cell, endothelial cell, adipocyte, smooth muscle
cell and cardiomyocyte. Using newly developed transgenic rat, we established
various endothelial cell lines from brain capillary, retinal capillary, bone
marrow, and choroid plexus, which can be used for a novel in vitro system to
investigate transport functions at the blood-tissue fluid barrier and to
develop high throughput screening systems. Our transgenic mouse and rat will be
useful to develop new types of cell lines with differentiation potentials. The
established cell lines will be useful to study tissue functions at cellular and
molecular levels and may be applicable to high throughput drug screening,
toxicity test, and to develop the alternatives to animal experiments.
Xiao-Yan Qiu*, Liang Zhang, Guo-Hui Li, Peng Lv,
Xiao-Hui Zhu, Peng Hao
( Peking
University Center for Human Disease Genomics, 38 Xue-Yuan Road, Beijing 100083,
China, *e-mail: qiuxy@bjmu.edu.cn )
Rearrangement, transcription, and expression of
immunoglobulin gene (Ig) were considered as B lymphocyte-specific events until
now. However, we have recently found that Ig expressed in many kinds of
epithelial tumor cells and overproliferation epithelium. In order to study Ig
gene expression in cancer cells and its significance, two cervical tumor cell
lines, HeLa S3 and HeLa MR, were used in the following experiments. 1) Ig
expression in HeLa S3 and HeLa MR. We identified IgG and IgM in two cell lines
by immunohistochemistry and FACs using anti-human IgG and IgM as antibodies.
Positive reaction, using anti-human IgG as antibody, was also found in culture
supernatant of both cell lines by ELISA. Furthermore, IgG expression was
identified in both cell lines by SDS-PAGE and Western blot. 2) Rearrangement of
Ig gene heavy chain variable region in HeLa S3 and HeLa MR. According to
immunology theory, V-D-J rearrangement of Ig gene does not occur in non-B
lymphocytes. In our study, V-D-J sequences of Ig gene in both cell lines
were detected by RT-PCR and three clones of each cell lines were analyzed by
DNA sequencing. The results demonstrated that Ig from HeLa S3 was monoclonal
while Ig from HeLa MR was not. Furthermore, Ig gene transcription
was proved by Northern blot using IgG1 Fc region as probe. 3) Anti-tumor
function of anti-human IgG in vitro and in vivo. In vitro
experiments showed that the apoptosis of cultured HeLa cells could be induced
by goat-anti-human IgG (approximately 20%-30%). Furthermore, we designed a test
to assess the effectiveness of anti-human IgG’s anti-tumor function in vivo.
The results demonstrated that anti-human IgG could significantly inhibit tumor
growth and development in vivo.
Jian Zhang, Xiao-Hui Zhu, Peng Hao, Yun-Tao Mao, Xin Sun, Le-Meng Wu, Guo-Hui Li, Xiao-Yan Qiu*
( Peking University
Center for Human Disease Genomics, 38 Xue-Yuan Road, Beijing 100083, China, *e-mail:
qiuxy@bjmu.edu.cn )
To our knowledge, immunoglobulins (Ig) are only
produced and secreted by differentiated B lymphocyte while other cells do not
express Ig because their Ig genes are in germline state. But recently, we found
a kind of protein, whose antigenicity was very similar to Ig, existed in some
malignant epithelial tumor cells (including primary and cultured cells) and in
some overproliferation epithelial cells. The object of this study was to
determine the Ig expression in lung cancer cells and to affirm a fact that
non-lymphocyte tumor cells may express Ig. We compared IgG expression in lung
cancer (n=12) to that in normal lung paracancer tissue (n=8)
using immmunohistochemistry (anti-human IgG as antibody) and in situ
hybridization (IgG1 Fc region cRNA as probe). The frequency of IgG expression
in cancer tissues was 11/12, while in paracancer normal tissues was 8/8 except
individual bronchia and alveolus epithelium cells. To determine IgG transcripts
in lung cancer (n=5) and normal paracancer tissue (n=5), Northern
blot, using probe of IgG1 Fc region, was performed. The result demonstrated
that the frequency of IgG transcripts in cancer was 5/5 and in normal tissues
was 2/5. The positive degree, however, in normal tissues was weaker than that
in cancer tissues. We also separated lung cancer cells from a case of lung
cancer tissue and removed lymphocytes. IgG from cancer cytoplasm was purified
by CM Sepharose Fast and determined by SDS-PAGE, Western blot, and N-terminal
amino acid sequencing analysis. After the concentration of purified IgG was
analyzed at ultraviolet 280 nm, 8%-10% of total proteins were estimated as IgG
and its purity was over 95% by Capillary Electrophoresis identification. These
results suggested that IgG might be overexpressed by epithelial tumor cells and
may be important for generation and proliferation of cancer cells.
Screening of
active vector planthoppers (Laodelphax striatellus) by selection and cross
breeding
( 1
Institute of Genetics, School of Life Sciences, Fudan University, Shanghai
200433, China; 2 College of Life Sciences, Qufu Normal
University, Qufu, Shandong 273165, P.R. China; *e-mail: zcqu@qfnu.edu.cn )
An active colony of Laodelphax striatellus, vector of
the rice stripe virus was obtained by cross breeding and selecting for four
successive generations and its transmitting ability was increased from 5.31% at
F0 generation to 25% in the fourth generation. When the colony was kept in a
free selection pressure for two years, the transmitting ability was reduced to
14.75%. Therefore, the selection pressure could play an important role in
maintaining vector transmission, no impact on insect acquisition feeding was
found. It was still detectable by dot immunobinding assay (DIBA) that
individual planthopper extraction was diluted into 1/30 and the acquired insect
rose from 10.82% in F0 generation to 32.6% in the fourth generation.
Role of spike
protein of rice ragged stunt oryzavirus in viral transmission by insect vector
Chao-Gang Shao, Jian-Hua Wu, Xiong-Bin Lu, Juan-Li Lei1, Den-Di Jin1, Shen-Xiang Chen1, Narayana M Upadhyaya2, Zu-Xun Gong*
( Key
laboratory of proteomics, Institute of Biochemistry and Cell Biology, Shanghai
Institute of Life Sciences, Academia Sinica, 320 Yue-Yang Road, Shanghai 20003,
China; 1The Virus Laboratory, Zhejiang Academy of Agricultural Sciences, 198
Shi-Qiao Road, Hangzhou 310021, China; 2CSIRO Plant Industry, GPO Box 1600,
Canberra, ACT, Australia; *e-mail: gongzx@sumn.shcnc.ac.cn )
Rice ragged stunt oryzavirus (RRSV) replicates in
both its insect vector-Nilaparvata lugens and plant host rice, and has a
complex multi-component particle bearing spikes on its outer surface. This 39
kD spike protein (P9), encoded by genomic segment 9 of RRSV, plays very
important role in the virus transmission by insect vector. Both feeding N.
lugens on purified E. coli expressed P9 and on transgenic rice
expressing the spike protein prior to infection of the rice by RRSV could
inhibit the vector transmission ability of the insect. The N. lugens was
significantly protected against RRSV infection after fed on P9. It was shown
that the viral titre in insects initially fed on transgenic plants and then on
diseased rice plants was inversely proportional to the levels of the spike
protein expressed in the transgenic plants. This suggests that P9 interferes
with the interaction between the intact virus particles and insect cell
receptors and the spike protein of RRSV probably contributes to vector
specificity. This approach would probably be a more environment-friendly and
sustainable method of virus control than by actual eradication of insect
vectors.
M R Jisnuson Svasti1,2*, Chantragan Srisomsap2, Rudee Surarit3, James Ketudat-Cairns4, Supanna Techasakul5
(1
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok
10400, Thailand;
2 Laboratory of Biochemistry, Chulabhorn
Research Institute, Bangkok 10210, Thailand;
3 Department of Physiology and Biochemistry,
Faculty of Dentistry, Mahidol University, Bangkok 10400, Thailand;
4 Institute of Science, Suranaree
University of Technology, Nakhon Ratchasima 30000, Thailand;
5Department of Chemistry, Faculty of
Science, Kasetsart University, Bangkok 10900, Thailand;
*e-mail:
scjsv@mahidol.ac.th )
Plant b-glucosidases (1.3.2.21) are a heterogeneous
group of enzymes, hydrolyzing various natural glycosides. They have potential
applications in biotechnology for synthesis of oligosaccharides by reverse
hydrolysis or alkyl glucosides by transglucosylation. We have compared the
synthesis reactions of cassava b-glucosidase (linamarase) specific for a
cyanogenic glucoside, Thai Rosewood b-glucosidase (dalcochinase) specific for
an isoflavonoid glucoside, and almond b-glucosidase specific for prunasin and
amygdalin.
Both dalcochinase and almond b-glucosidase give good
yields of disaccharides and trisaccharides in reverse hydrolysis, but
linamarase shows little capability for reverse hydrolysis. Almond enzyme can also
use fucose for reverse hydrolysis, but dalcochinase has little ability to use
fucose. Optimum pH and temperature for reverse hydrolysis were similar to
hydrolysis, and glucose helps to stabilise dalcochinase at high temperature.
Dalcochinase can also synthesise alkyl glucosides from p-nitrophenyl-glucoside
and alkyl alcohols, and can catalyse reactions with longer chain alcohols
better than almond glucosidase. Primary alcohols are better acceptors than
secondary alcohols, while tertiary alcohols cannot react. Linamarase, however,
is the only enzyme that can use tertiary alcohols as acceptors in
transglucosylation. Thus, despite their structural similarities, the
b-glucosidases show interesting differences in their substrate specificity for
the hydrolysis, reverse hydrolysis and transglucosylation reactions, so they
are useful models for study of structure-function relationships.
This work was supported by the National Research
Council of Thailand, Chulabhorn Research Institute, and Thailand Research Fund.
Asia’s chance in modern biology
(IMCB, Singapore, *e-mail:
yht@imcb.nus.edu.sg )
This year’s A-IMBN meeting in Shanghai is an opportunity
to review the building of biological sciences in Asia and evaluate the
opportunities for A-IMBN to create opportunities for Asian scientists to be key
players where biology meets the physical sciences. I will discuss whether the
historical science “lag” in Asia might indeed become an advantage to create
unique opportunities for us to build now.
( Department
of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science,
3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, *e-mail:
kichihar@rinshoken.or.jp
)
There are growing lines of evidence addressing the
importance of the ubiquitin-proteasome system that regulates the cell cycle,
immune response, signalling cascades and developmental programs. This
proteolysis machinery also plays a principal role in selective destruction of
misfolded or impaired proteins generated in the cell. Here, I discuss our
current knowledge of structure, functions, and pathophysiology linked to the
ubiquitin-proteasome pathway.
The HIV-1
translational frameshift site: A site of potential antiviral therapy
Warren P Tate*,
Elizabeth S Poole, Ryan Graves
( Department
of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand, *e-mail:
hod.biochemistry@stonebow.otago.ac.nz )
HIV-1 continues to be a devastating epidemic,
particularly in the developing world, where combination anti-retroviral therapy
(HAART) has proven too expensive to be readilyaccessible to affected
individuals. Simpler, lower cost alternative drugs or complementary therapies
are desperately needed.
HIV-1 uses a unique translational recoding feature (programmed -1 frameshift) to produce an exact ratio of different proteins (gag & gag-pol) from the same RNA sequence. Within the human genome, only genes for antizyme, involving in regulating polyamines, have been conclusively confirmed to use a frameshift mechanism in their expression, but it is a forward +1 shift, rather than a backwards shift, making the –1 frameshift event of HIV-1 a potential drug target.
We originally characterised the HIV-1 frameshift
mechanism on bacterial ribosomes and proposed a new model,
“post-translocational slippage”. In this model, a “slippery sequence” (U UUU
UUA) in the HIV-1 RNA, is positioned on the ribosome in the post
translocational state with a GGG codon for glycine in the empty tRNA binding
site. Two sites for binding tRNA at the active centre of the ribosome (P;
peptidyl & E; exit sites) are occupied, while the third site (A-aminoacyl
site) awaits the incoming tRNA. We have now tested this model on mammalian
ribosomes in cell culture.
A reporter system was constructed containing the
HIV-1 frameshift sequence or modifications of it. We replaced the A site codon
in the HIV-1 RNA in our model with a stop codon so that instead of a GGG codon
(predicted to be at the A site from our model) there would be a UGA whose
decoding could be manipulated. This caused a decrease in frameshift efficiency
that was further sensitive in vivo to over-expression of eukaryotic
decoding factor (eRF1). This implies the mammalian ribosome is in the
post-translocational state prior to slippage, consistent with our proposed model.
Revealing the exact state of the ribosome during
slippage, and the mechanism of the –1 frameshifting, provides an opportunity
for the choice of specific drugs that would target the ribosome in that state.
Proof of concept has been achieved in vitro in 2 of 8 drugs tested where the –1
frameshifting event could be selectively affected at concentrations of the
drugs that did not affect overall protein synthesis, or +1 frameshifting with
the human antizyme sequence. A selection of drugs is now being tested in
vivo with cultured cells. Such drugs may be able to perturb the ratio of
viral proteins by influencing the frameshifting specifically without affecting
the other cognate events of protein synthesis. If this could be utilised to
upset the balance of HIV proteins and thereby formation of infective particles
there is hope that this might represent another low cost drug to keep the viral
titres at manageable levels.
Novel genes
cloned from a neuronal cell line newly established from a cerebellum of an adult
p53-/- mouse
( Department
of Biological Science and Technology, Tokyo University of Science, 2641
Yamazaki, Noda, Chiba 278-8510, Japan, *e-mail: tomoylab@rs.noda.tus.ac.jp )
The neurons of the mammalian central nervous system are highly specialized in function and morphology. They are generated from neuroepithelial cells of a neural tube, and are differentiated to a large number and variety of neurons. Many genes are expected to be involved in the irreversible process from undifferentiated to differentiated status. We attempted to isolate such genes from a cell line 2Y-3t newly established from a mouse cerebellum. We took a subtraction method to isolate genes expressed differerentially in differentiated cells, and 17 cDNA clones were isolated. Functions of 6 cDNA clones are unknown. No.60 cDNA clone has 723 nucleotides encoding 240 amino acid residues. It contains two putative EF-hand motifs and a coiled-coil region at C terminal end. A recombinant full-length No.60 protein had Ca2+ binding activity as determined by a Ca2+-induced mobility shift in SDS-PAGE. Expression of the clone was undetectable at embryonic stage and was increased in brain during development. In situ hybridization showed that the expression was observed predominantly in neurons. We developed antisera against No. 60 protein to determine the subcellular localization during differentiation of 2Y-3t cells. They cease proliferation and become morphologically neurons in serum-free medium, in which expression of nestin is decreased, while NF-L expression is increased. In differentiated 2Y-3t cells, No.60 protein was localized in the cytoplasm, particularly concentrated at tips of neurites. We analyzed functions of No. 60 protein by overexpressing the protein in undifferentiated 2Y-3t cells expressing a low endogenous level of the protein. Overexpression of the protein led to extension of neurite-like process in undifferentiated 2Y-3t cells. These results suggest that No.60 gene may play roles in the neuronal morphology.
ERK/MAKP
signaling in cancer cells and its new binding protein, Naf1(Nef associated
factor 1) which attenuates the EGF/ERK2 nuclear signaling
Nobuo Tsuchida1*, S L Zhang1, T Fukushi1, L Huang1, R Arvind1, S Ren1, Y Kaneda1,2, K Omura2, F Jin1, N Yamamoto3, K Koike4
( 1Dept.
of Molecular Cellular Oncology and Microbiology; 2Dept. of
Oral Surgery; 3Dept. of Microbiology, Graduate School, Tokyo
Medical & Dental University, Tokyo Japan 113-8549; 4Dept.
of Gene Research, Cancer Instittue (JFCR), Tokyo Japan 170; *e-mail:
tsuchida.mcom@tmd.ac.jp )
ERK1/2 is an important factor in signal transduction,
controls cell growth, differentiation and death. To elucidate roles of ERK1/2
signaling in the genesis of human cancer, we examined 15 head and neck squamous
cell carcinoma cell lines for the amplifications of ERK2 locus by CGH
and FISH, and found two of them to have amplified DNA copy in this region.
Accordingly, we compared ERK1/2 mRNA levels, the presence of ras mutations and
EGFR amplification levels. Although 10 of them had one of these abonormalities,
only one cell line had both H-ras mutaiton and EGFR amplification. Further, we
examined expression levels of these proteins in EGF/ERK signaling pathway in
colorectal tumors by Western blotting. There was a good correlation of
expression levels of EGFR, H-ras, MEK, and ERK1/2. Taken together, these
results suggest the activation of the signal transducdtion pathway of ERK1/2 to
be important in tumorigenesis.
Activated ERK1/2
is translocated from the cytoplasm into the nucleus where transcription factors
involved in cell growth and differentiation are activated. We have identified
new ERK2-interacting proteins by using yeast two hybrid system. One of them was
Naf1 HIV Nef-associated factor 1. This protein was confirmed to bind to ERK2 in
human cells by pull down assay in vitro and immunoprecipitation in vivo,
and to be a substrate of ERK phohsphorylation. However, this protein was found
to block nuclear signaling of ERK2 by trapping in the cytoplasm. It is
suggested that Naf1 could be a new type of attenuator of ERK2 activation
signaling, as it is also a substrate of ERK2. Further experiments how EGF/ERK
signal modulates HIV Nef protein function are being done.
Development of
nested PCR for the rapid detection and identification of Salmonella typhi
( 1United
Laboratories, Inc. Mandaluyong City, Philippines; 2 Research
& Biotechnology Division, St. Luke’s Medical Center, Philippines; *e-mail:
elena-tuano@yahoo.com or rrm@skyinet.net )
A nested PCR based on the flagellin gene sequence was
developed to detect Salmonella typhi, the causative agent of typhoid fever. The
S. typhi flagellin sequences were downloaded from GenBank, NCBI. Based on the
conserved sequences, two pairs of oligonucleotide primers were designed using
the DNAsis and Oligo 4.1 software to amplify a 370 bp fragment of the flagellin
gene of S. typhi. Amplified prodeucts were visualized by agarose gel
electrophoresis followed by ethidium bromide staining. Fifty-eight S. typhi and
fifty-eight non S. typhi organisms obtained from the bacterial collection of
the Microbiology Section, Biological Science Department, Medical Affairs
Division of United Laboratories, Inc. from 1996-2000 were tested using the
nested PCR. The recommended nested PCR using the newly designed primers was
found to be highly specific and highly sensitive. Currently, the developed
method is being applied on clinical samples collected from patients.
From gene to
therapy: Growth factor receptors as targets for the development of smart drugs
( Max-Planck-Institute
for Biochemistry, Martinsried 82152, Germany, *e-mail: ullrich@biochem.mpg.de )
Cancer represents a disease prototype that is
connected to defects in the cellular signal transduction network that controls
proliferation, motility, survival and recognition by the immune system. The
spectrum of genetic alterations identified in cancer cells includes mutations
in various genes leading to structural and functional dysfunctions in signal
transmission and definition as well as over- or underexpression of positive or
negative signal generating or regulating proteins respectively. For the past
years we have investigated various aspects of signalling systems in tumor cells
in order to identify critical switchpoints in the pathophysiological process
that results in malignancy. These efforts aim at the selective blockage of
abnormal, disease-promoting signaling mechanisms rather than the eradication of
all growing cells in the body as in in the case of currently used
chemotherapeutics and began with the cloning of EGF receptor cDNA and the
related receptor HER2/neu. Recently the work that began in 1983 yielded the
first specific oncogene-based therapeutic “Herceptin” for the treatment of
mammary carcinoma. Analogous “target-driven drug development” efforts have led
to the identification of a germ line mutation of FGFR4(4FGF receptor 4-gene)
which predisposes the carrier for a more aggressive progression of breast
cancer, which emphasizes the potential value of the FGFR4 as target for the
development of anti-metastatic therapeutics. Moreover through the
identification of a crosstalk mechanism between G protein- coupled receptors
and members of the EGFR family, new cancer relevant targets have been
identified that are upstream of the EGFR and include membrane-standing
metalloproteases. In addition to the investigation of genetic alterations
involved in cancer development and metastasis we have characterized Flk-1/VEGFR2
as a critical signaling element in tumor angiogenesis which represents the
basis for the development of novel anti-angiogenic therapies for a broad
spectrum of cancers.
Mass
spectrometry as a tool to study effects of phosphorylation on actin-binding
properties of caldesmon
( Muscle and
Motility Group, Boston Biomedical Research Institute 64 Grove St., Watertown,
Massachusetts 02472, USA, *e-mail: wang@bbri.org )
Protein phosphorylation is a fundamental mechanism
for intracellular signal transduction. To elucidate the signaling pathways, it
is essential to determine both the level and the site(s) of phosphorylation of
proteins involved. Traditionally, this is achieved by using radioactive
isotopes (γ-32P-ATP). Although highly
sensitive, this approach fails to reveal phosphorylation prior to the addition
of hot ATP. The other newer approach is by specific antibodies against
phosphopeptides. However, one must first know the phosphorylation sites, and
only phosphorylation at these sites can be detected. Matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry serves as another effective
means to detect covalent modifications such as phosphorylation on proteins. We
have applied this method to the study of smooth muscle caldesmon (CaD), which
inhibits actomyosin interactions through actin binding and regulates smooth
muscle contraction. CaD is phosphorylated in vivo by extracellular signal
regulated kinases (ERKs) at Ser759 and Ser789, but the functional consequence
of such a modification has not been clear. Nor has there been any clear data
showing ERK-induced phosphorylation significantly changes the affinity of CaD
for F-actin. We have examined this issue by mass spectrometry coupled with
enzyme digestion and actin co-sedimentation. Our data indicate that
actin-binding segments of CaD containing Ser759 and Ser789 bind actin much more
weakly after ERK phosphorylation, whereas other actin-binding segments are not
affected by ERK treatment. This finding not only explains the lack of evidence
thus far for a clear difference in actin-binding of full-length CaD upon ERK
phosphorylation, but also provides a plausible mechanism for the regulatory
action of ERKs on CaD and other substrates. This study further demonstrates
that for a protein of known sequence, MALDI-mass spectrometry combined with
limited proteolysis serves as a useful tool both to detect phosphorylation and
to identify the phosphorylation sites.
Type IA DNA
topoisomerases: A key role in the maintenance of genome stability
( Department
of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts
02138, USA, *e-mail: jcwang@fas.harvard.edu )
Among the four subfamilies of DNA topoisomerases, the
type IA enzymes are unique in their omnipresence: all bacteria, eukarya, and
archea examined to date are found to possess one or more type IA enzymes. In
mammals, two of the six known DNA topoisomerases, DNA topoisomerase IIIa and
IIIb, are of the type IA subfamily. Targeted gene disruption experiments in the
mouse model showed that inactivation of IIIa leads to early embryonic death,
and inactivation of IIIb leads to shortened lifespan.
Both mammalian enzymes, similar to Escherichia
coli and yeast DNA topoisomerase III, possess a rather weak activity in the
relaxation of negative supercoils. Thus their functional importance is
particularly surprising in view of the presence of the type IB and IIA DNA
topoisomerases, which are robust in supercoil removal. Recent studies in E.
coli and yeast suggest that the type IA DNA topoisomerases play a key role
in the processing of a DNA structure, or structures, that are formed in
recombination/repair. From the known mechanisms of different subfamilies of DNA
topoisomerases, it has been suggested that the type IA enzymes are specifically
involved in the resolution of a recombination intermediate termed a double
Holliday junction. Whereas other cellular activities, such as the resolvases,
are capable of processing these structures, only the type IA DNA topoisomerases
are expected to resolve them in a way that leads to no crossing-over. For mice
lacking DNA topoisomerase IIIb, in addition to a shortened lifespan the animals
also suffer a gradual decrease in fertility over time and through generations,
suggesting an accumulation of chromosomal defects. Examination of meiotic cells
showed a high frequency of aneuploidy, which can be interpreted in terms of a
key role of type IA DNA topoisomerases in the resolution of the double Holliday
junction.
Identification
of PDCD5, a mammalian protein that promotes apoptosis by binding caspase-3 and
XIAP
Ying Wang&,
Xianting Li&, Rong-Hua Sun, Ying-Yu Chen, Quan-Sheng Song,
Ying-Mei Zhang, Da-Long Ma*