Myosin Light Polypeptide Research Articles

The uterine smooth muscle S‐nitrosylproteome in pregnancy and functional effects of S‐nitrosoglutathione

The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not known. Nitric oxide relaxes uterine smooth muscle in a cGMP independent manner. S‐Nitrosylation, the covalent addition of an NO‐group to a cysteine thiol is a likely mechanism to explain the ability of NO to relax myometrium. For the first time we describe the myometrial S‐nitrosylproteome in pregnant and non‐pregnant guinea pig and the human labor, not in labor, and pre term laboring comprehensive proteome and S‐nitrosylproteome using multidimensional protein identification technology. We show that S‐nitrosylation of the intermediate filament protein desmin is significantly increased (5.7 fold, p < 0.005) in pregnancy and that this increase cannot be attributed solely to the increase in protein expression (1.8 fold, p < 0.005) that accompanies pregnancy. We also show that both myosin light regulatory polypeptide 9 and myosin light chain kinase are disparately S‐nitrosylated in the different states of pregnancy. We further describe both the S‐nitrosylation and activation of the potassium channel TREK‐1 by the NO donor S‐nitroso‐glutathione. Elucidation of the myometrial proteome and S‐nitrosylproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction‐relaxation.

Proteomics of a new esophageal cancer cell line established from Persian patient

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study.

Proteomic analysis of pubocervical fascia in women with and without pelvic organ prolapse and urodynamic stress incontinence

This pilot study compares protein expression patterns in the pubocervical fascia of women with pelvic organ prolapse (POP) and stress urinary incontinence (SUI) and asymptomatic women with normal pelvic support. Samples of pubocervical fascia were collected from four women with POP and SUI and from three asymptomatic (control) women. These were analyzed using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The expression levels of transgelin, smooth muscle gamma-actin, myosin light polypeptide 6, and alpha-1 antitrypsin precursor were more than twofold higher in patients than in controls. An additional five proteins were overexpressed (more than twofold) in patients, while three proteins were detected only in the patients. This pilot study is the first study to estimate changes in protein expression in the pubocervical fascia of human patients with POP. These changes could be related to the pathophysiology of POP.

Identification of novel transcripts from the porcine MYL1 gene and initial characterization of its promoters

The fast skeletal alkali myosin light polypeptide 1 (MYL1) gene is one of three mammalian alkali MLC genes and encodes two isoforms, 1f and 3f, which play a vital role in embryonic, fetal, and adult skeletal muscle development. We isolated the MYL1 gene from a pig BAC library with the goal of characterizing its promoter and identifying its transcripts. Genes and isoforms were identified by reverse transcriptase-PCR, northern blot and RACE (Rapid Amplification of cDNA Ends). Potential MYL1 gene promoters were characterized using a luciferase reporter assay and electrophoretic mobility shift assays (EMSA). MLC1f, MLC3f, and three additional isoforms of porcine MYL1, MLC5f-A, -B, and -C were identified. Up to now, the three novel isoforms had not been reported in human or mouse. Northern blot analysis indicated that MLC1f, MLC3f, and MLC5fs were expressed only in longissimus dorsi muscles. Two transcription initiation and termination sites were identified by RACE. Promoter analysis and EMSA demonstrated the presence of a MEF3 (skeletal muscle-specific transcriptional enhancer) binding site (+384 to +481), which might be essential for porcine MYL1 transcription. Our results suggested that five transcript variants were generated using alternative promoters, two transcription start sites, and polyA sites, as well as variable splicing of the pig MYL1 exon 5. The identification of alternative promoters and splice variants, the expression of the splice variants in different muscle tissues, and the definition of regulatory elements provide important molecular genetic knowledge concerning the MYL1 gene.

Comparative proteomic analysis of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-transformed and normal human bronchial epithelial G0/G1 cells

In the present study, we investigated the proteomic profiling of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE)-transformed human bronchial epithelial cell line (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. Differential analysis of proteomic profiling indicated that 67 polypeptides were down-regulated and 77 polypeptides were up-regulated in 16HBE-C G0/G1 cells compared to 16HBE G0/G1 cells. Then 16 differentially expressed protein spots were analyzed with Q-TOF MS/MS. Of these spots, 3 down-regulated polypeptides were identified as sorcin, small ubiquitin-related modifier 2 precursor and eukaryotic translation initiation factor 5A-1, and 9 up-regulated polypeptides were identified as calmodulin, myosin light polypeptide 6, eukaryotic translation initiation factor 6, proliferating cell nuclear antigen (PCNA), tumor protein D52 (TPD52), superoxide dismutase [Cu-Zn], prohibitin, nuclear protein Hcc-1 and vimentin. These proteins are involved in cell proliferation, protein synthesis, signal transduction and carcinogenesis. Western blotting analysis verified the increased expression levels of PCNA and TPD52 in 16HBE-C G0/G1 cells. Based on the clues from proteomic analysis, the migration and invasion capabilities of 16HBE-C and 16HBE cells were tested. The results indicated that 16HBE-C cells showed much higher migration and invasion capabilities than 16HBE cells, and moreover, the suppression of TPD52 by RNAi resulted in significant decrease of migration and invasion capabilities of 16HBE-C cells. These results will be valuable for further investigating and understanding the mechanisms underlying BaP-induced carcinogenesis.

Down-Regulation of 14-3-3 Isoforms and Annexin A5 Proteins in Lung Adenocarcinoma Induced by the Tobacco-Specific Nitrosamine NNK in the A/J Mouse Revealed by Proteomic Analysis

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in the A/J mouse model. Here we identified and validated, using two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry and immunoblotting, proteins that are differentially expressed in the lungs of mice treated with NNK versus vehicle control treatment. We also determined whether protein levels in the lungs of NNK-treated mice could be further modulated by the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC). The proteins identified in this study are SEC14-like 3, dihydropyrimidinase-like 2, proteasome subunit alpha type 5, annexin A5, 14-3-3 protein isoforms (theta, epsilon, sigma, and zeta), Rho GDP dissociation inhibitor alpha, myosin light polypeptide 6, tubulin-alpha-1, vimentin, Atp5b protein, alpha-1-antitrypsin, and Clara cell 10 kDa protein (CC10). Among those proteins, we demonstrated for the first time that 14-3-3 isoforms (theta, epsilon, and sigma) and annexin A5 were significantly down-regulated in mouse lung adenocarcinoma induced by NNK and were recovered by p-XSC. These proteins are involved in a variety of biological functions that are critical in lung carcinogenesis. Identification of these proteins in surrogate tissue in future studies would be highly useful in early detection of lung adenocarcinoma and clinical chemoprevention trials.

Microarray study of gene expression profiles in peripheral blood samples from lung cancer patients before and after erlotinib treatment

e19072 Background: The gene expression profile in peripheral blood samples from lung cancer patients is a potential predictor to treatment response. Methods: The study has been developed using 10 healthy volunteers as the control group and 10 lung cancer patients (stage IV). Written informed consent was obtained being the protocol approved by the local Clinical Research and Ethics Committee. Peripheral blood samples were obtained from lung cancer patients before (T0) and after treatment (T15d). RNA from peripheral blood samples was extracted and purified selecting 28S/18S ratios>1.5 to obtain cDNA and cRNA for hybridization of the 20,000 genes included in Human 20K CodeLink. An array from each participant was obtained in duplicate. For each array, 2 μg of cRNA was compared to 2 μg of healthy cRNA.. Significant genes were found using Significance Analysis of Microarrays which uses repeated permutations of the data. Results: The selected genes were expressed >3-fold with a false discovery rate =0.05. Before treatment (T0) when patients were compared to healthy volunteers there was an increase in the expression of: histone 1 H4c, transforming growth factor beta 2, endothelial cell growth factor 1 (platelet-derived), glucose-6-phosphatase catalytic 2, Relaxin 3 receptor 1, Insulin-like growth factor binding protein 2, RAS-like family 11 member B, and ELK4. After treatment (T15d), when each lung cancer patient's results were compared to their own before treatment results (T0), there was an increase in the expression of: Bcl2, myosin light polypeptide 4; interferon alpha-inducible protein 27; interferon gamma receptor 1; RASSF5, ARHGEF6, IGFBP5, tumor protein p53 inducible nuclear protein 1, peroxisome proliferative activated receptor gamma. Conclusions: The data presented identifies biologically relevant over-expressed genes in lung cancer. A validation of these results and the analysis of the genes that identify patients who will respond positively to erlotinib treatment is being carried out. No significant financial relationships to disclose.

Ecstasy produces left ventricular dysfunction and oxidative stress in rats

Our aim was to determine whether the repeated, binge administration of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) produces structural and/or functional changes in the myocardium that are associated with oxidative stress. Echocardiography and pressure-volume conductance catheters were used to assess left ventricular (LV) structure and function in rats subjected to four ecstasy binges (9 mg/kg i.v. for 4 days, separated by a 10 day drug-free period). Hearts from treated and control rats were used for either biochemical and proteomic analysis or the isolation of adult LV myocytes. After the fourth binge, treated hearts showed eccentric LV dilation and diastolic dysfunction. Systolic function was not altered in vivo; however, the magnitude of the contractile responses to electrical stimulation was significantly smaller in myocytes from rats treated in vivo with ecstasy compared with myocytes from control rats. The magnitude of the peak increase in intracellular calcium (measured by Fura-2) was also significantly smaller in myocytes from ecstasy-treated vs. control rats. The relaxation kinetics of the intracellular calcium transients were significantly longer in myocytes from ecstasy-treated rats. Ecstasy significantly increased nitrotyrosine content in the left ventricle. Proteomic analysis revealed increased nitration of contractile proteins (troponin-T, tropomyosin alpha-1 chain, myosin light polypeptide, and myosin regulatory light chain), mitochondrial proteins (Ub-cytochrome-c reductase and ATP synthase), and sarcoplasmic reticulum calcium ATPase. The repeated binge administration of ecstasy produces eccentric LV dilation and dysfunction that is accompanied by oxidative stress. These functional responses may result from the redox modification of proteins involved in excitation-contraction coupling and/or mitochondrial energy production. Together, these results indicate that ecstasy has the potential to produce serious cardiac toxicity and ventricular dysfunction.

Proteomic analysis of selenium embryotoxicity in cultured postimplantation rat embryos

Developmental toxicity of selenium (Se) is a nutritional, environmental and medicinal concern. Here, we investigated Se embryotoxicity by proteomic analysis of cultured rat embryos. Rat embryos at day 9.5 or 10.5 of gestation were cultured for 48 or 24 h, respectively, in the presence of sodium selenate (100 or 150 microM) or sodium selenite (20 or 30 microM). Proteins from the embryo proper and yolk sac membrane were analyzed by two-dimensional electrophoresis for quantitative changes from those in control embryos. Proteins with quantitative changes were identified by mass spectrometric analysis. Growth inhibition and morphological abnormalities of cultured embryos were observed in all the Se treatment groups. By the analysis of the embryo proper, actin-binding proteins were identified as proteins with quantitative changes by selenate: increased phosphorylated-cofilin 1, increased phosphorylated-destrin, decreased drebrin E, and decreased myosin light polypeptide 3. Many proteins showed similar changes between selenate and selenite, including increased ATP-synthase, decreased acidic ribosomal phosphoprotein P0, and decreased pyrroline-5-carboxylate reductase-like. In the yolk sac membrane, antioxidant proteins were identified for protein spots with quantitative changes by selenite: increased peroxiredoxin 1 and increased glutathione S-transferase. The identified proteins with quantitative changes by selenate or selenite were considered to be candidate proteins involved in Se embryotoxicity: the actin-binding proteins for selenate embryotoxicity, proteins with the similar changes for the common Se embryotoxicity and antioxidant proteins for modification of Se embryotoxicity by redox-related treatments. These proteins may also be used as biomarkers in developmental toxicity studies.

Mechanisms of acrolein-induced myocardial dysfunction: implications for environmental and endogenous aldehyde exposure

Aldehydes are ubiquitous pollutants generated during the combustion of organic materials and are present in air, water, and food. Several aldehydes are also endogenous products of lipid peroxidation and by-products of drug metabolism. Despite well-documented high reactivity of unsaturated aldehydes, little is known regarding their cardiovascular effects and their role in cardiac pathology. Accordingly, we examined the myocardial effects of the model unsaturated aldehyde acrolein. In closed-chest mice, intravenous acrolein (0.5 mg/kg) induced rapid but reversible left ventricular dilatation and dysfunction. In mouse myocytes, micromolar acrolein acutely depressed myofilament Ca(2+) responsiveness without altering catecholamine sensitivity, similar to the phenotype of stunned myocardium. Immunoblotting revealed increased acrolein-protein adducts and protein-carbonyls in both acrolein-exposed myocardium (1.8-fold increase, P < 0.002) and myocytes (6.4-fold increase, P < 0.02). Both the contractile dysfunction and adduct formation were markedly attenuated by pretreatment with the thiol donor N-acetylcysteine (5 mM). Two-dimensional gel electrophoresis and mass-assisted laser desorption/ionization time-of-flight mass spectrometry analysis revealed two groups of adducted proteins, sarcomeric/cytoskeletal proteins (cardiac alpha-actin, desmin, myosin light polypeptide 3) and energy metabolism proteins (mitochondrial creatine kinase-2, ATP synthase), indicating site-specific protein modification that was confirmed by immunohistochemical colocalization. We conclude that direct exposure to acrolein induces selective myofilament impairment, which may be, in part, related to the modification of proteins involved in myocardial contraction and energy metabolism. Myocardial dysfunction induced by acrolein and related aldehydes may be symptomatic of toxicological states associated with ambient or occupational exposures or drug toxicity. Moreover, aldehydes such as acrolein may mediate cardiac dysfunction in pathologies characterized by high-oxidative stress.

Hedgehog-independent overexpression of transforming growth factor-β1 in rhabdomyosarcoma of Patched1 mutant mice

The tumor suppressor gene PATCHED1 (PTCH1) is a member of the hedgehog signaling pathway and causatively associated with several human sporadic and familial cancers, including those of the skin, muscle and brain. Inactivation of one Ptch1 allele in the mouse results in the development of medulloblastoma and rhabdomyosarcoma (RMS), the latter being a malignant tumor of skeletal muscle origin. To identify genes involved in the pathogenesis of Ptch1-associated RMS, we have monitored the expression of 588 genes in RMS and normal skeletal muscle (SM) of heterozygous Ptch1neo67/+ mice using cDNA array technology. RMS displayed increased transcript levels of several genes such as transforming growth factor-beta1 (Tgfb1), insulin-like growth factor 2 (Igf2), villin 2 (Vil2), integrin beta1 (Itgb1), Sloan-Kettering viral oncogene homolog (Ski), and insulin-like growth factor binding protein 3 (Igfbp3), as well as numerous genes coding for structural components of myogenic cells such as myosin light polypeptide 4 (Myl4), myosin light polypeptide 6 (Myl6), and vimentin (Vim). Detailed promoter analysis revealed a putative Gli binding site in the second promoter region (P2) of the murine Tgfb1 gene. However, using reporter assay we show that the P2 promoter is not responsive to hedgehog signaling. We furthermore describe that Tgfb1 expression could not be activated in C2C12 myoblasts in the presence of murine Shh-N peptide and that Tgfb1 is equally expressed in both wild-type and Ptch1-deficient mouse embryos. In line with this, TGFB1 was strongly expressed in human RMS cell lines independently of the GLI1 expression status. In summary, our results suggest that aberrant expression of Tgfb1 may be involved in RMS development in a way that is independent of hedgehog signaling.

Consumption of a moderately Zn-deficient and Zn-supplemented diet affects soluble protein expression in rat soleus muscle

Zinc deficiency negatively affects muscle function, but there are limited biochemical data identifying the cause of this reduction in function. The objective of the present study was to identify soluble proteins in rat soleus muscle that were responsive to different levels of dietary zinc. Rats ( n=21) were fed diets containing three concentrations of zinc: 5, 30 and 200 ppm for 42 days. There was no difference in body weights of the rats consuming the 5-ppm zinc diet compared to the rats consuming the 30- or 200-ppm zinc diets; however, bone zinc levels were significantly decreased in the 5-ppm dietary zinc group. Individual soluble protein fractions were isolated from these muscles and the samples were prepared for two-dimensional polyacrylamide gel electrophoresis. The expression levels of four proteins were significantly depressed by dietary Zn depletion and supplementation, S-glutathiolated carbonic anhydrase, myosin light polypeptide 3, heat shock protein 20 and heart fatty acid binding protein. This is the first report that indicates that both Zn depletion and supplementation result in protein expression profiles that may negatively affect skeletal muscle function. These results indicate that there are specific signaling pathways that require proper Zn nutriture for maintaining optimal muscle function and suggest that the consumption of pharmacologic doses of Zn may be detrimental to muscle function.

Methylation Status of Genes Upregulated by Demethylating Agent 5-aza-2′-Deoxycytidine in Hepatocellular Carcinoma

Background/Aims: To determine the clinical significance of gene promoter methylation in hepatocellular carcinoma (HCC), we examined in clinical samples the methylation status of those promoters that showed elevated activity in hepatoma cell lines after 5-aza-2′-deoxycytidine treatment. Methods: Regarding the genes with promoter hypermethylation in the cell lines, their expression levels and methylation status in HCC and non-HCC tissues were assessed by semiquantitive RT-PCR and methylation-specific PCR. To confirm the result, the expression levels and methylation status in 16 additional HCC and non-HCC tissues were assessed. Results: The promoter regions of caveolin 1 (CAV1), cysteine and glycine-rich protein 1 (CSRP1), Kruppel-like factor 6 (KLF6), myosin (light polypeptide 9) (MYL9), and transgelin (TAGLN) were highly methylated in the cell lines. CAV1 and CSRP1 were methylated in HCC more frequently than in non-HCC. KLF6, MYL9, and TAGLN were fully methylated in both HCC and non-HCC. Using additional clinical samples, downregulation of CAV1 and CSRP1 was observed in 38 and 56%, respectively, of the 16 HCC samples and aberrant methylation of CAV1 and CSRP1 was observed in 56% of HCC in both cases. Conclusion:CAV1 and CSRP1 were inactivated in HCC by aberrant methylation and they may serve as important biomarkers of malignancy.

Proteome Mapping of the Effects of Age, Pressure Overload, and Aldosterone Antagonism on Cardiac Fibrosis

Aging, pressure overload, and aldosterone signaling have each been implicated as causes of cardiac fibrosis. Cardiac fibrosis, an excessive accumulation of extracellular matrix, promotes diastolic dysfunction and congestive heart failure. Treatment with spironolactone (SP), an aldosterone antagonist, is effective in attenuating cardiac fibrosis, but questions remain regarding the mechanisms of action and the efficacy of aldosterone antagonism in advanced age. PURPOSE: The purpose of the present study was to identify the independent and combined effects of age, aortic constriction, and aldosterone antagonism on the cardiac proteome. METHODS: Female F344 rats served as untreated controls (C) or were subjected to aortic constriction (AC) at 5 (Y) or 23 (O) months of age and treated with SP (30 mg/kg/day) or vehicle (V) for 6 months, yielding the following six groups: Y-C, Y-V, Y-SP, O-C, O-V, O-SP. Left ventricle free walls (LV) were harvested and quick-frozen in liquid nitrogen and were subsequently used to generate 2-dimensional electrophoretic (2-DE) proteome maps. PDQuest software was used to analyze SYPRO Orange-stained 2-DE gels, quantify spot densities, and compare expression levels between groups. Protein spots were identified with mass spectrometry of trypsin digests. Significance Analysis of Microarrays (SAM) was used to evaluate the protein expression levels, and a false discovery rate of <1% was used as a guideline to identify differentially expressed proteins. RESULTS: Proteins differentially expressed as a function of age included Immt-Er (−25%), dihydrolipoamide acetyltransferase (+173%), albumin (+40 to +64%), ATP synthase mitochondrial F0 complex (−39%), isovaleryl CoA dehydrogenase (−15%), acetyl-CoA dehydrogenase long-chain (−13%), creatine kinase (−20 to −33%), gluathione S-transferase (−44%), and ATP synthase mitochondrial F1 complex (+865%). Aortic constriction caused changes in lactate dehydrogenase B (+73%) and myoglobin (+73%). Spironolactone induced changes in myosin light polypeptide 3 (−53), albumin (−23 to −55%), isovaleryl CoA dehydrogenase (−34%), malate dehydrogenase (+42%), and creatine kinase (+30%). CONCLUSIONS: Aging and pressure overload are associated with a marked change in the stoichiometry of a wide range of metabolic proteins. Spironolactone treatment had mixed effects, exacerbating the age-associated decrease of some metabolic proteins, but increasing levels of other metabolic proteins. Supported by NIH R03 AG022625 and the Undergraduate Research Opportunity Program at the University of Michigan

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.

Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer

To assess the proteome of normal versus tumor tissue in squamous cell carcinoma of the esophagus (SCCE) in Iranian patients and compare our results with former reports by using proteomics. Protein was extracted from normal and tumor tissues. Two dimensional electrophoresis was carried out and spots with differential expression were identified with mass spectrometry. RNA extraction and RT-PCR along with immunodetection were performed. Fourteen proteins were found whose expression levels differed in tumor compared to normal tissues. Mass spectrometric analysis resulted in the identification of beta-tropomyosin (TMbeta), myosin light chain 2 (and its isoform), myosin regulatory light chain 2, peroxyredoxin 2, annexin I and an unknown polypeptide as the down regulated polypeptides in tumor tissue. Heat shock protein 70 (HSP70), TPM4-ALK fusion oncoprotein 2, myosin light polypeptide 6, keratin I, GH16431p and calreticulin were the up-regulated polypeptides found in tumor tissue. Several of these proteins, such as TMbeta, HSP70, annexin I, calreticulin, TPM4-ALK and isoforms of myosins, have been well recognized in tumorigenesis of esophageal or other types of cancers. Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCE, including TMbeta.

CD24 and myosin light polypeptide 2 are involved in prevention of experimental autoimmune encephalomyelitis by myelin basic protein-pulsed dendritic cells

We previously demonstrated that injection of myelin basic protein-pulsed (MBP-pulsed)–but not of unpulsed–autologous bone marrow-derived dendritic cells (DC) efficiently prevents experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To define the molecules involved, we used 3 groups of rats pretreated subcutaneously with MBP-DC, or unpulsed DC, or PBS (control EAE). Four weeks later, all rats were immunized with encephalitogenic MBP peptide and adjuvant. Microarray analyses were done to screen for genes that differ among the 3 groups. Based on microarray analysis data, we used real-time PCR to measure expression of six probably involved genes in draining lymph node cells obtained on day 0, day 7 and day 14 post immunization (p.i.). Two of these 6 genes were consistently altered in both microarray analyses and RT-PCR. They are CD24 antigen being persistently low, and myosin light polypeptide 2 (Myl2) being high in the acute immune response in MBP-DC pretreated rats that develop resistance to EAE. These two genes could be targeted to treat EAE and, possibly, multiple sclerosis.

Functional proteome of bones in rats with osteoporosis following ovariectomy

Osteoporosis is a chronic condition chiefly affecting postmenopausal women, in whom the skeleton loses a significant percentage of its mineralized mass and mechanical resiliency, thereby becoming prone to fracture. Although the effect of the loss of estrogen on bone metabolism has been documented, its mechanism is still poorly understood. In the present proteomic study, we characterized the effect of estrogen deficiency on protein expression in rat bones. Using two-dimensional gel electrophoresis, mass spectrometry and rat protein database, we successfully identified three distinctly changed proteins named thioredoxin peroxidase 1, myosin light polypeptide 2 and ubiquitin-conjugating enzyme E 2-17 kD, among which ubiquitin-conjugating enzyme E 2-17 kD has been documented to be an estrogen-related protein, but the other two are first reported to be osteoporosis-related proteins in the current study. These results provide valuable experimental evidences for the elucidation of the molecular mechanism of osteoporosis related to the loss of estrogen.

Linkage assignment of the fast skeletal alkali myosin light polypeptide 1 (MYL1) gene to porcine chromosome 15.

Linkage assignment of the fast skeletal alkali myosin light polypeptide 1 (MYL1) gene to porcine chromosome 15.

Cloning of a Parathyroid Hormone/Parathyroid Hormone-Related Peptide Receptor (PTHR) cDNA from a Rat Osteosarcoma (UMR 106) Cell Line: Chromosomal Assignment of the Gene in the Human, Mouse, and Rat Genomes

Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acylpeptide hydrolase gene (APEH). Our results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes.