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 Pdf file on Expression of cytosolic
Katarzyna Lechward* and Kinga Tkacz-Stachowska
Department of Molecular Enzymology, Intercollegiate Faculty of
Biotechnology, Medical
*Correspondence address. Tel: t48-58-3491470;
Fax: t48-58-3491445; E-mail: [email protected]
Our previous studies had shown that cytosolic
Keywords ATP metabolism;
cytosolic
Received: November 18, 2008 Accepted: February 11, 2009
Introduction
ATP metabolism is crucial for energetic homeostasis of every living cell
and its balancing is of primary importance of surviving and maintaining the
sense of wellbeing [1,2]. Several mechanisms based on positive and negative
feedback loops are implicated into regulation of this balance, including a key
player adenosine. Adenosine is produced by the group of enzymes called We repeatedly observed that in so-called red, slow-twitch, oxidative
muscles, the expression of both cN-I RNA and protein was noticeably higher than
in so-called fast-twitch, white glycolytic ones. On the basis of that
observation, we hypothesized that there could be a correlation between
expression of cN-I and the type of metabolism in different muscles. We decided to put this
hypothesis under the tests and screen human muscle samples for the expression
of cN-I type A and B as well as marker of oxidative metabolism-myoglobine [6].
Materials and Methods
Chemicals
Ammonium persulfate, Ficoll 400, bromophenol blue, and sodium citrate were
from SERVA Electrophoresis (Human muscle samples
Fragments of human muscles were obtained from the Department of Emergency
Surgery at Northern blot analysis
Total cellular RNA was isolated from muscle samples by the method of
Chomczynski and Sacchi [7] based on acid guanidinium thiocyanate–phenol–chloroform extraction. Northern blot analysis was routinely performed on
10–25 mg of total RNA as described previously [5]. Intensities of signals were
quantified by Sigma Scan software and statistical analysis was performed with
Statistica 6.0 software.
SDS–PAGE and western
blot analysis
Human muscle fragment homogenates were prepared as described previously
[8], and as standard we assayed 150 mg of protein on SDS–PAGE followed by transfer onto nitrocellulose membrane. Dilutions of the
antibodies were as follows: 1:100 anti-cN-I (kindly provided by Graciela
Sala-Newby from Bristol Heart Institute,
Results
Classification of muscle samples according to cN-I gene
and protein expression
To verify expression levels of cN-I, total RNA was isolated from all
samples according to the method described earlier. The procedure yielded
between 42 and 231 mg RNA with appropriate OD260/280 value (1.7–2.1). In the first
approach of the study, we classified the muscle samples according to the expression
levels of cN-I gene and
cN-I protein by northern and western blot techniques, respectively. Six types
of muscles were chosen: m. gluteus maximus, m. quadriceps femoris, m. tibialis anterior, m. brachoradialis, m. deltoideus, and m. biceps brachii. All muscle samples, except for m. quadriceps femoris, originated from 10 healthy men of the
similar age (22–31 years), living
active, sporty lifestyle, and undergoing the bone fracture-related treatments.
The number of patients is smaller than the number of samples collected due to
several cases of multiple limbs fractures.
The patients were subjected to surgery due to different injuries such as
serious limbs breakage, internal fixation, or replacement of fracture. m. quadriceps
femoris was
taken from a 38-year-old man during total hip replacement operation. Results
were confirmed by running each RNA sample at least twice and performing
hybridization procedures under the same experimental conditions. Figure 1 shows the
representative northern blot analysis of RNA isolated from m. gluteus
maximus, m. quadriceps
femoris, m. tibialis
anterior, m. brachoradialis, m. deltoideus, and m. biceps brachii, with 18S RNA serving
as loading control. Human cN-I gene expression was found to vary in different muscles, with the highest
in m. quadriceps
femoris and m. gluteus
maximus and the
lowest in m. deltoideus and m. brachoradialis.
Our next question was whether the levels of RNA encoding cN-I corresponded
to actual amount of protein present in the tissues, as one could expect
different posttranscriptional control mechanisms operating there. Therefore, we
performed immunoblotting on high-speed cytoplasmic muscle extracts with
antibodies against human cN-I (Fig. 2) and human cN-I A and B isoformspecific antibodies (Fig. 3). As shown in Fig. 2, antihuman cN-I
antibodies recognized major band of ~46 kDa. Two smaller
and less intense bands of a mass of 42 and 30 kDa were most likely to be
proteolytic fragments on cN-I. Antigen competition experiments proved that all
of the bands were specific (data not shown). When one compared the levels of
RNA and protein for single sample, it can be seen that the amount of RNA is
proportional to the amount of proteins. Using the recombinant cN-I protein of
the known mass (47 kDa) as a standard, the molecular weight of human muscle
cN-I could be estimated at the same level. The relative amounts of protein
correlated with observed mRNA levels in every skeletal muscle sample tested,
were the most significant in m. quadriceps femoris and m. gluteus maximus and the lowest in m. deltoideus and m. brachoradialis. Major conclusion
taken from Fig. 3(A) was that the isoform expressed in tested samples was cN-I A. We tried to
assay the expression of cN-I B isoform by both, Western blot analysis [Fig. 3(B)] and RT–PCR approach (data not shown), but we obtained no signals from any of the
samples tested. The protein of ~47 kDa was detected
only with hcN-I A antibodies in all three muscle samples. This emphasizes the
suggestion that the only cN-I isoform expressed in human skeletal muscles is
hcN-I A. Since the prevailing type of muscle tissue which we obtained was m. quadriceps
femoris, we
focused on those samples and decided to do more detailed statistical analysis
of correlation between cN-I expression and oxidative metabolism.
Classification of m.quadriceps femoris muscle samples
according to myoglobine gene expression The level of oxidative metabolism in 38
samples of m. quadriceps femoris was assessed by quantifying the amount of myoglobine transcript. Myoglobine,
despite some controversies found in the literature, was one of the most
relevant markers of oxidative fibers and we took it as a reference for our
screen. We assayed muscle samples that originated from highly homogenous group
of patients in terms of disability (total hip replacement), age (69–74), and gender (slight prevailing men: 57.9%). Taking into account the
type of performed surgery and the age of patients, we assumed that their
physical activity was rather low with the periods of immobilization. Tested
muscle samples showed significant variation in respect of myoglobine message
levels. We had quantified the intensities of signals by means of Sigma Scan
software and standardized it with the expression of 18S RNA gene. Based on that
quantification, we created four groups characterized by none (2), moderate (t), high (tt), and very high (ttt) myoglobine (Table 1) and cN-I (Table 2) genes expression in
38 quadriceps
femoris muscle
fragments from a group of patients of different sex (57.9% of men) in the age
ranging from 69 to 74 years. The same set of data was used in Fig. 4, yet Tables 1 and 2 show how the values
were distributed in the population, whereas Fig. 4 delineates what is the real relation
between the measured values and ideal correlation curve. Figure 4 shows the correlation
plot between myoglobine and cN-I gene expression levels. The P-value was calculated
as 0.508, what showed that there was no relationship between expression levels
of cN-I and myoglobine in human muscles. There was little doubt that cN-I was
expressed independently of the level of oxidative metabolism in 38 human quadriceps
femoris muscle
fragments.
Discussion
To our knowledge, this is the first attempt to correlate and quantify the
expression of cN-I, engaged in ATP catabolism with fiber composition of human
skeletal muscles. Our studies showed that the predominant form of cytosolic Following the initial observation about the uneven distribution of cN-I
gene and protein in fast- and slow-twitch muscles, we undertook the work in
humans, but we did not incorporate so-called behavioral and physiological
differences between both species. The major locomotion mechanism utilized by
pigeons is an active flight, and they cross very short distances by walking,
when compared with humans whose main way of locomotion is walking. Humans, in
general, use the whole array of skeletal muscle to keep the vertical posture
during locomotion, yet they have very diverse life style, which includes
different levels of daily exercises, the walked distances, and the usage of
mechanical engine-based means of transportation tools in the urban zones.
Acknowledgements
We would like to thank our colleagues, surgeons from the Department of
Emergency Surgery Medical
Funding
Molecular biology reagents including enzymes, except Klenow polymerase
were purchased from grant from Polish State Committee for Research no PO406015
for KL, and the rest of reagents were purchased from overhead costs of the
Laboratory of Molecular Enzymology. Klenow polymerase was kind gift of Dr
Joanna Jakubkiewicz-Banecka, from Department of Molecular Biology of
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