Myosin Alkali Light Chain Research Articles

Myosin light chain of shark fast skeletal muscle exhibits intrinsic urea-resistibility

Marine elasmobranch fish contain urea, a protein denaturant, in their bodies. The urea-trimethylamine N-oxide (TMAO) counteraction mechanism contributes to urea-resistibility, where TMAO compensates for protein denaturation by urea. However, previous studies revealed that shark fast skeletal muscle myosin exhibits native activity at physiological urea concentrations in the absence of TMAO, suggesting that shark myosin has urea-resistibility. In this study, we compared the urea-resistibility of myosin alkali light chains (A1-LC and A2-LC) from banded houndshark and carp by examining the α-helical content at various urea concentrations. The α-helical content of carp myosin A1-LC and A2-LC gradually decreased as urea concentrations increased to 2 M. In contrast, the α-helical content of banded houndshark A1-LC increased between 0 and 0.5 M urea, and the α-helical content of A2-LC remained constant until 0.5 M urea. We determined the full-length sequences of the banded houndshark myosin light chains (A1-LC, A2-LC and DTNB-LC). Hydrophilicity analysis revealed that the N-terminal region (residues 28–34) of A1-LC from banded houndshark is more hydrophilic than the corresponding region of A1-LC from carp. These findings support the notion that shark myosin exhibits urea-resistibility independent of the urea-TMAO counteraction mechanism.

Identification of Schistosoma japonicum GSK3β interacting partners by yeast two-hybrid screening and its role in parasite survival.

Schistosoma is the causative agent of schistosomiasis, a common infectious disease distributed worldwide. Our previous phosphoproteomic analysis suggested that glycogen synthase kinase 3 (GSK3), a conserved protein kinase in eukaryotes, is likely involved in protein phosphorylation of Schistosoma japonicum. Here, we aimed to identify the interacting partners of S. japonicum GSK3β (SjGSK3β) and to evaluate its role in parasite survival. Toward these ends, we determined the transcription levels of SjGSK3β at different developmental stages and identified its interacting partners of SjGSK3β by screening a yeast two-hybrid S. japonicum cDNA library. We further used RNA interference (RNAi) to inhibit the expression of SjGSK3β in adult worms in vitro and examined the resultant changes in transcription of its putative interacting proteins and in worm viability compared with those of control worms. Reverse transcription-quantitative polymerase chain analysis indicated that SjGSK3β is expressed throughout the life cycle of S. japonicum, with higher expression levels detected in the eggs and relatively higher expression level found in male worms than in female worms. By screening the yeast two-hybrid library, eight proteins were identified as potentially interacting with SjGSK3β including cell division cycle 37 homolog (Cdc37), 14-3-3 protein, tegument antigen (I(H)A), V-ATPase proteolipid subunit, myosin alkali light chain 1, and three proteins without recognized functional domains. In addition, SjGSK3β RNAi reduced the SjGSK3β gene transcript level, leading to a significant decrease in kinase activity, cell viability, and worm survival. Collectively, these findings suggested that SjGSK3β may interact with its partner proteins to influence worm survival by regulating kinase activity.

MUSCLE DEVELOPMENT IN AMPHIOXUS: MORPHOLOGY, BIOCHEMISTRY, AND MOLECULAR BIOLOGY

ABSTRACT This review concerns the structure and biochemistry of muscle in amphioxus. Most work has focused on the segmented swimming (axial) muscles. These muscles derive from the medial wall of the somites, which arise as evaginations from the gut wall. The myotomal muscle cells of amphioxus, unlike those of vertebrates, never fuse, but remain mononucleate, contain only one myofibril, and span the entire length of the myotome. The muscle cells are very thin and lack a T-tubule system. There are two, maybe three, types of fibers. Innervation is via muscle tails, which contact the basal lamina of the nerve cord. The notochord is also composed of striated muscle cells, which similarly send muscle tails to the nerve cord. Less is known about the biochemistry of muscle. The notochord, like molluskan catch muscle, contains paramyosin. Among the muscle-specific proteins sequenced are alkali myosin light chain, troponin C and sarcoplasmic calcium-binding proteins, calcium-vector protein, and its target protein c...

Proteomic research on anti-atherosclerosis mechanism of curcumin in RAW264.7

Two-dimensional difference gel electrophoresis and mass spectrum were used to study the anti-atherosclerosis mechanism of curcumin. The proteins from RAW264.7 cell and RAW264.7 cell treated with 25 micromol x L(-1) curcumin were labeled with Cy3 or Cy5 randomly. Each Cy3-labeled sample and Cy5-labeled sample was mixed on the same 2-D gel along with a Cy2-labeled mixture of all samples as an internal standard and run on the same gel. The gels were scanned under different wave-length light after electrophoresis. All images were analyzed by DeCyder 6.5 software, and the different proteins were identified by mass spectrum. The expression of ATP synthesis H+ transporting, MHC class II, non-muscle myosin alkali light chain and cytochrome b5 increased in the RAW264.7 cell treated with 25 micromol x L(-1) curcumin, while the expression of phosphodiesterase 4D, elF-3, Hnrpf protein, vimentin, nucleophosminl and Ranbp 1 decreased. The anti-atherosclerosis mechanism of curcumin is the result of enhancement of the cell inflammation, antioxidant activity and inhibition of cholesterol transport, reduce of the accumulation of intracellular cholesterol and other factors. In addition, curcumin also had the effect of anti-tumor through regulated tumor cell differentiation and apoptosis.

Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity

In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac β-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rβ-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1)and human ventricular (hVLC-1)light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or β-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rβ-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

BackgroundRegulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken.ResultsWe have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines.ConclusionsFrom these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species.

Isolation, characterization and comparison of Atlantic and Chinook salmon growth hormone 1 and 2

BackgroundGrowth hormone (GH) is an important regulator of skeletal growth, as well as other adapted processes in salmonids. The GH gene (gh) in salmonids is represented by duplicated, non-allelic isoforms designated as gh1 and gh2. We have isolated and characterized gh-containing bacterial artificial chromosomes (BACs) of both Atlantic and Chinook salmon (Salmo salar and Oncorhynchus tshawytscha) in order to further elucidate our understanding of the conservation and regulation of these loci.ResultsBACs containing gh1 and gh2 from both Atlantic and Chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions. These BACs also contain the genes for skeletal muscle sodium channel oriented in the same direction. The sequences of the genes for interferon alpha-1, myosin alkali light chain and microtubule associated protein Tau were also identified, and found in opposite orientations relative to gh1 and gh2. Viability of each of these genes was examined by PCR. We show that transposon insertions have occurred differently in the promoters of gh, within and between each species. Other differences within the promoters and intronic and 3'-flanking regions of the four gh genes provide evidence that they have distinct regulatory modes and possibly act to function differently and/or during different times of salmonid development.ConclusionA core proximal promoter for transcription of both gh1 and gh2 is conserved between the two species of salmon. Nevertheless, transposon integration and regulatory element differences do exist between the promoters of gh1 and gh2. Additionally, organization of transposon families into the BACs containing gh1 and for the BACs containing gh2, are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between the gh1- and gh2-containing paralogous BACs for the two fish species. This is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to Atlantic and Pacific salmon. The Chinook and other Oncorhynchus GH1s are strikingly different in comparison to the other GHs and this change is not apparent in the surrounding non-coding sequences.

Integration of embryonic and fetal skeletal myogenic programs at the myosin light chain 1f/3f locus

The genetic control of skeletal muscle differentiation at the onset of myogenesis in the embryo is relatively well understood compared to the formation of muscle during the fetal period giving rise to the bulk of skeletal muscle fibers at birth. The Mlc1f/3f ( Myl1) locus encodes two alkali myosin light chains, Mlc1f and Mlc3f, from two promoters that are differentially regulated during development. The Mlc1f promoter is active in embryonic, fetal and adult fast skeletal muscle whereas the Mlc3f promoter is upregulated during fetal development and remains on in adult fast skeletal muscle. Two enhancer elements have been identified at the mammalian Mlc1f/3f locus, a 3′ element active at all developmental stages and an intronic enhancer activated during fetal development. Here, using transgenesis, we demonstrate that these enhancers act combinatorially to confer the spatial, temporal and quantitative expression profile of the endogenous Mlc3f promoter. Using double reporter transgenes we demonstrate that each enhancer can activate both Mlc1f and Mlc3f promoters in vivo, revealing enhancer sharing rather than exclusive enhancer–promoter interactions. Finally, we demonstrate that the fetal activated enhancer contains critical E-box myogenic regulatory factor binding sites and that enhancer activation is impaired in vivo in the absence of myogenin but not in the absence of innervation. Together our observations provide insights into the regulation of fetal myogenesis and the mechanisms by which temporally distinct genetic programs are integrated at a single locus.

Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat

This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

Molecular characterization of a myosin alkali light chain-like protein, a “concealed” antigen from the hard tick Haemaphysalis qinghaiensis

There should be some differences between antibodies generated by feeding ticks on animals and those derived by immunizing animals with tick extracts. Here, we found serum collected from sheep immunized with Haemaphysalis qinghaiensis salivary gland extracts could detect two more protein bands with molecular weights of 22 and 37 kDa (P22 and P37) on Western blots of extracts of tick salivary glands than serum from tick infected animals. Rabbit anti- H. qinghaiensis differential protein immune serum was then generated from P22 and P37 and was used to immunoscreen a cDNA library constructed from salivary glands, Malpighian tubules and ovaries of partially engorged H. qinghaiensis. A cDNA contains an open reading frame of 483 bp that codes for 160 amino acid residues with a coding capacity of 18 kDa was cloned and designated Hq02. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other organs and different developmental stages of H. qinghaiensis. The predicted amino acid sequence of the Hq02 gene had high homology to some known myosin alkali light chain (MLC) proteins. A fusion protein consisting of 130 amino acids of Hq02 protein and 335 amino acids of T7 gene 10 protein was expressed in Escherichia coli and used to immunize sheep. Western blot showed that only rabbit anti- H. qinghaiensis differential protein immune serum could recognize the expressed Hq02 protein, while rabbit anti- H. qinghaiensis saliva immune could not. This proved Hq02 protein was a “concealed” antigen. Immunization with the recombinant Hq02 conferred a 21.8% reduction of engorgement weight for adult female ticks that fed on the immunized sheep. This is the first report of tick myosin alkali light chain and the function of this protein is discussed.

Dependence of cross‐bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres

Cross-bridge kinetics underlying stretch-induced force transients was studied in fibres with different myosin light chain (MLC) isoforms from skeletal muscles of rabbit and rat. The force transients were induced by stepwise stretches (< 0.3% of fibre length) applied on maximally Ca2+-activated skinned fibres. Fast fibre types IIB, IID (or IIX) and IIA and the slow fibre type I containing the myosin heavy chain isoforms MHC-IIb, MHC-IId (or MHC-IIx), MHC-IIa and MHC-I, respectively, were investigated. The MLC isoform content varied within fibre types. Fast fibre types contained the fast regulatory MLC isoform MLC2f and different proportions of the fast alkali MLC isoforms MLC1f and MLC3f. Type I fibres contained the slow regulatory MLC isoform MLC2s and the slow alkali MLC isoform MLC1s. Slow MLC isoforms were also present in several type IIA fibres. The kinetics of force transients differed by a factor of about 30 between fibre types (order from fastest to slowest kinetics: IIB > IID > IIA >> I). The kinetics of the force transients was not dependent on the relative content of MLC1f and MLC3f. Type IIA fibres containing fast and slow MLC isoforms were about 1.2 times slower than type IIA fibres containing only fast MLC isoforms. We conclude that while the cross-bridge kinetics is mainly determined by the MHC isoforms present, it is affected by fast and slow MLC isoforms but not by the relative content of MLC1f and MLC3f. Thus, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the cross-bridge kinetics.

Multidimensional Liquid Chromatography Separation of Intact Proteins by Chromatographic Focusing and Reversed Phase of the Human Serum Proteome: OPTIMIZATION AND PROTEIN DATABASE

In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation (undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic "cellular" proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present.

Nerve influence on myosin light chain phosphorylation in slow and fast skeletal muscles

Neural stimulation controls the contractile properties of skeletal muscle fibres through transcriptional regulation of a number of proteins, including myosin isoforms. To study whether neural stimulation is also involved in the control of post-translational modifications of myosin, we analysed the phosphorylation of alkali myosin light chains (MLC1) and regulatory myosin light chains (MLC2) in rat slow (soleus) and fast (extensor digitorum longus EDL) muscles using 2D-gel electrophoresis and mass spectrometry. In control rats, soleus and EDL muscles differed in the proportion of the fast and slow isoforms of MLC1 and MLC2 that they contained, and also in the distribution of the variants with distinct isoelectric points identified on 2D gels. Denervation induced a slow-to-fast transition in myosin isoforms and increased MLC2 phosphorylation in soleus, whereas the opposite changes in myosin isoform expression and MLC2 phosphorylation were observed in EDL. Chronic low-frequency stimulation of EDL, with a pattern mimicking that of soleus, induced a fast-to-slow transition in myosin isoforms, accompanied by a decreased MLC2 phosphorylation. Chronic administration (10 mg x kg(-1) x d(-1) intraperitoneally) of cyclosporin A, a known inhibitor of calcineurin, did not change significantly the distribution of fast and slow MLC2 isoforms or the phosphorylation of MLC2. All changes in MLC2 phosphorylation were paralleled by changes in MLC kinase expression without any variation of the phosphatase subunit, PP1. No variation in MLC1 phosphorylation was detectable after denervation or cyclosporin A administration. These results suggest that the low-frequency neural discharge, typical of soleus, determines low levels of MLC2 phosphorylation together with expression of slow myosin, and that MLC2 phosphorylation is regulated by controlling MLC kinase expression through calcineurin-independent pathways.

On the alkali light chains of vertebrate skeletal myosin. Nucleotide binding and salt-induced conformational changes.

The interaction of alkali light chains of vertebrate skeletal myosin with nucleotides and KCl has been examined by chemical modifications of these proteins, by direct binding measurements, and in circular dichroism studies. The reactivities of the single thiol groups in the isolated alkali light chains A1 and A2 have been studied by reacting these proteins with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). MgATP and MgADP reduced the reactivities of thiol groups while high concentrations of KCl increased them. Subsequent equilibrium dialysis experiments verified the presence of a low-affinity nucleotide binding site per each alkali subunit. Circular dichroism measurements revealed that KCl induced local (around phenylalanines) and overall (alpha-helical content) conformational changes of equal magnitude in the two alkali light chains. However, salt induced different conformational changes in the subfragment 1 isoenzymes, S-1(A1) and S-1(A2). This differential salt effect on the S-1 isoenzymes was confirmed by comparing their thermal stability in different salt conditions. AT low KCl concentrations (5 mM), S-1(A1) was found to be considerably more heat labile than S-1(A2); at higher salt levels (50 mM KCl) the stability of S-1(A1) approached that of S-1(A2). These experiments are discussed in terms of the relationship between the alkali subunits and the ATP and the actin-binding sites of myosin.

Distinct gene signatures of transient and acute megakaryoblastic leukemia in Down syndrome.

Approximately 10% of newborns with Down syndrome develop Transient Leukemia (TL), a disorder that is unique to infants with constitutional trisomy 21 (or trisomy 21 mosaicism). TL blasts disappear spontaneously within the first 3 months of life in the majority of cases. Despite the resolution of TL, 20-30% of these newborns will go on to develop acute megakaryoblastic leukemia (AMKL) later in life. In this study, samples from both TL and AMKL patients were examined using cDNA microarrays to study the pathogenic progression from TL to AMKL. TL and AMKL samples partition separately by cluster analysis, and AMKL samples had substantial increases in apolipoprotein C-I, transporter 1, myosin alkali light chain 4, and spermidine/spermine N-acetyltransferase, compared to TL samples. Although these findings will require validation in an independent series of TL and AMKL samples, they indicate that TL and AMKL have distinct gene signatures, and provide a basis for studies of the different mechanisms underlying either the resolution of TL or its progression to AMKL.

Endurance training-induced changes in alkali light chain patterns in type IIB fibers of the rat

The effects of endurance training on the expression of myosin were electrophoretically analyzed in the deep portion of vastus lateralis muscle from the rat. A 10-wk running program led to increases (P < 0.01) in myosin heavy chain (MHC) 2a and 2d with a decrease (P < 0.01) in MHC(2b). Training also evoked a rearrangement of the isomyosin pattern with decreases in fast isomyosin (FM) 1 (P < 0.01) and FM2 (P < 0.05) and a rise in intermediate isomyosin (P < 0.01). These changes were accompanied by a 61% decrease (P < 0.01) in myosin light chain (MLC) 3F (11.8 +/- 2.7 vs. 4.6 +/- 4.2%). Two-dimensional electrophoresis made it possible to separate the triplet of isomyosins (FMb) consisting of MHC(2b). Training elicited a 26% decrease (P < 0.05) in the FM1b fraction within FMb, i.e., FM1b/(FM1b + FM2b + FM3b) (24.2 +/- 5.5 vs. 18.0 +/- 4.3%). These changes resulted in a 10% decrease (P < 0.05) in the MLC(3F) fraction, i.e., MLC(3F)/(MLC(1F) + MLC(3F)), in FMb (44.9 +/- 4.5 vs. 40.3 +/- 3.2%). These results suggest that endurance training may exert the depressive effect on the contractile velocity of type IIB fibers and that a training-induced decrease in the contractile velocity of whole muscle may be caused by alterations in fast alkali MLC complements within a given fiber type as well as by transitions in MHC-based fiber populations.

Characterization of gene expression profile associated with energy restriction-induced cold tolerance of heart.

Hypothermia is known to be a common feature of energy restriction (ER) and essential for a life-prolonging effect of ER. The heart is sensitive to hypothermia, but the heart in ER mice acquires some adaptation to hypothermia. The aim of the present study was to characterize the gene expression profile associated with ER-induced cold resistance of heart. We analyzed the expression of heart mRNA from ER (200 kJ/week) or control (400 kJ/week) B6 11-month-old male mice using cDNA array membranes including 588 genes. Eighty-eight out of 588 genes were expressed in the heart. mRNAs increased by ER were glutathion S-transferase Mu1, transcriptional factor 1 for heat shock gene (HSF1), and fetal myosin alkali light chain genes. mRNA decreased by ER were seven genes in four categories: (1). cell cycle or apoptosis-related proteins (cyclin G and nucleoside diphosphate kinase B); (2). stress response proteins (oxidative stress-induced protein); (3). DNA repair proteins (protein involved in DNA double-strand break repair, Rad23 UV excision repair protein homologue and ubiquitin-conjugating enzyme); and (4). cell-surface antigens (lamimin receptor 1). These data suggest that the heart of ER mice adapts to hypothermia involving heat shock proteins and their transcriptional factors and by changing structure and property of myofibrils. It is also suggested that ER induces protection against oxidative stress and inhibits cell proliferation of "nonmuscle cells" in the heart. Gene expression analysis using cDNA array was useful for screening genes associated with ER-induced cold adaptation.

Thyroid hormone gene targets in ROS 17/2.8 osteoblast-like cells identified by differential display analysis.

Thyroid hormone plays an important role in bone development and metabolism. We used a polymerase chain reaction (PCR)-based mRNA differential display (DD) analysis to obtain a profile of thyroid hormone-responsive genes in osteoblast-like cells (ROS 17/2.8). ROS 17/2.8 cells were treated with 10(-8) M triiodothyronine (T(3)) for 2 and 24 hours. Total RNA was isolated, reverse-transcribed, and amplified using a total of 72 combinations (2 hours) and 240 combinations (24 hours) of 5' and 3' primers. At the 2-hour time point, 1 true-positive novel clone was identified and shown to be the mitochondrial gene, subunit 6 of ATP synthase (ATPase-6). At the 24-hour time point, 3 differentially expressed (DE) mRNAs were confirmed as true-positives including; nonmuscle alkali myosin light chain (NM aMLC), ATPase-6, and one novel clone. T(3)-induction of ATPase-6 mRNA in ROS 17/2.8 cells was seen at 2 and 4 hours, but was maximal at 24 hours (2.1-fold). T(3) induction of ATPase-6 mRNA was increased to fourfold in ROS 17/2.8 cells cultured at a low density. NM aMLC mRNA was modestly upregulated by T(3) in ROS 17/2.8 cells by 1.4-fold, and induction was augmented at low cell density to 1.7-fold. T(3) action on NM aMLC and on the mitochondrial gene ATPase 6, represent novel targets and potential mediators of thyroid hormone action on bone. Cell type, and the extent of cell differentiation, influences T(3) regulation of genes in osteoblast-derived cells.

Up-regulation of transcription of smooth muscle myosin alkali light chain by ethanol in human breast cancer cells.

Both epidemiological and experimental studies indicate that ethanol is a tumor promoter and that chronic ethanol exposure enhances metastasis of breast cancer cells, and with an in vitro model (T47D human breast cancer cells), we have previously demonstrated that ethanol exposure stimulated the migration of breast cancer cells. In the present study, differential display reverse transcription polymerase chain reaction was used to identify ethanol-responsive genes in T47D cells. Three differentially displayed, ethanol-responsive gene fragments were identified, and their expression was confirmed by Northern blot hybridization. Sequence analysis revealed that one cDNA fragment represented the myosin alkali light chain (MLC 1sm) of human smooth muscle. The expression of MLC 1sm was found to be significantly higher in breast cancer cells than in normal mammary epithelial cells. With T47D cells, ethanol induced an additional duration- and concentration-dependent up-regulation of MLC 1sm. At 400 mg/dl, an ethanol-mediated increase was evident at 6 h (55% increase), peaked at 24 h (2.7-fold increase) following exposure, and diminished thereafter. At pharmacologically relevant concentrations (e.g., 100 mg/dl), ethanol produced a significant increase of MLC 1sm expression, and progressively higher ethanol concentrations resulted in more up-regulation. The half-life of MLC 1sm mRNA was not altered, however, the transcription rate of MLC 1sm was significantly increased by ethanol. MLC is a structural component of the cytoskeleton of eukaryotic cells, and it plays critical roles in the regulation of cell shaping, movement, and growth. Thus, ethanol-mediated up-regulation of MLC may be an underlying molecular mechanism for its tumor promoting effect.

Developmental program expression of myosin alkali light chain and skeletal actin genes in the rainbow trout Oncorhynchus mykiss

We have isolated MLC1 F (tMLC1 F), MLC3 F (tMLC3 F) and skeletal actin cDNAs from the teleost Oncorhynchus mykiss. Sequence analysis indicates that tMLC1 F and tMLC3 F are not produced from differentially spliced mRNAs as reported in avians and rodents but are encoded by different genes. Results from RNase protection analysis showed that the corresponding transcripts are expressed in fast skeletal muscles. Whole-mount in situ hybridisation revealed distinct expression patterns of the myosin alkali light chains and skeletal actin genes during skeletal muscle development in the embryo.