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Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas), and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586 bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas, which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In mammals, four tropomyosin genes TPM1, TPM2, TPM3, and TPM4 are known. One isoform of the TPM3 gene, encoding 285 amino acid residues designated as TPM3α, has been reported. TPM3α protein expression in human hearts is not definitively established. We have cloned from human heart and skeletal muscle transcripts of TPM3α and three novel TPM3 isoforms, TPM3ν, TPM3ξ, and TPM3ο. TPM3ν and TPM3ο are alternatively spliced RNAs with different 3'-UTRs encoding an identical novel protein with 285 amino acid differing from TPM3α and TPM3ξ in exon 6 only. TPM3α and TPM3ξ, which have different 3'UTRs, also encode an identical protein. qRT-PCR data show that the transcripts of TPM3α, TPM3ν, TPM3ξ, and TPM3ο are expressed in both heart and skeletal muscle. We have evaluated the expression of various TPM proteins in fetal and adult human hearts, and also in skeletal muscle samples. Western blots using CG3 antibody show a stronger signal of TPM3 protein in fetal heart and adult skeletal muscle compared to adult heart. LC-MS/MS studies with the protein spots separated and identified by CH1 antibody after 2D Western blot analyses, confirm the expression of TPM3α/TPM3ξ in heart, but some peptides detected could be either TPM3α or TPM3ν. In heart samples, TPM1 protein was the dominant with varying amount of TPM2 and TPM3, while TPM4 expression was not observed. In skeletal muscles, TPM2 was the majority TPM protein expressed. The biological consequences of these varying expression of individual tropomyosin proteins are yet to be established., (© 2020 Wiley Periodicals LLC.)

Objective: The aim of this study was to elucidate the role of TPM4 in the progression of hepatocellular carcinoma (HCC), and to explore the potential underlying mechanism by interacting with SUSD2., Patients and Methods: TPM4 expression levels in 41 HCC tissues and paracancerous tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between TPM4 level with the pathological indexes and overall survival of HCC patients was analyzed. TPM4 overexpression and knockdown models were constructed in Bel-7402 and Hep3B cells, respectively. Subsequently, Cell Counting Kit-8 (CCK-8) and transwell assay were conducted to assess the effects of TPM4 on the proliferative and migratory abilities of HCC cells. Dual-Luciferase reporter gene assay was performed to verify the binding relationship between TPM4 and SUSD2. In addition, the xenograft model was conducted in HCC-bearing mice administrated with Hep3B cells in vivo. Finally, the effect of TPM4 on the growth of HCC was explored., Results: TPM4 was significantly upregulated in HCC tissues and cell lines. Higher rates of lymphatic and distant metastasis, as well as worse prognosis, were observed in HCC patients with higher expression level of TPM4. The overexpression of TPM4 significantly enhanced the viability and migration abilities of Bel-7402 cells. However, opposite results were observed after the knockdown of TPM4 in Hep3B cells. SUSD2 was verified to be the target of TPM4 and was negatively regulated by TPM4. SUSD2 was lowly expressed in HCC tissues and cell lines. Meanwhile, SUSD2 was considered to be responsible for TPM4-regulated progression of HCC. In mice administrated with Hep3B, the cells transfected with sh-TPM4, the tumor volume and weight of HCC were markedly reduced when compared with the controls., Conclusions: TPM4 level is correlated with high rates of lymphatic and distant metastasis, as well as poor prognosis of HCC patients. By negatively targeting SUSD2, TPM4 aggravates the progression of HCC by accelerating the proliferative and migratory abilities of HCC cells.

Background: Tropomyosin alpha-1 chain (TPM1) is a member of the tropomyosin family and the expression of TPM1 is found to be dysregulated in various tumors. The present study aimed to investigate the clinical performance and significance of TPM1 in gastric cancer., Methods: First, the levels of TPM1 mRNA and protein were detected through real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) respectively. The correlation between TPM1 expression and clinicopathological variables was analyzed. Then, receiver operating characteristic (ROC) curve was applied to determine the diagnostic performance of TPM1 in gastric cancer. Finally, overall survival analysis was carried out using Kaplan-Meier method in order to determine the prognostic performance of TPM1 in gastric cancer., Results: Compared with the controls, TPM1 mRNA and protein expression levels were significantly downregulated in patients with gastric cancer. Downregulation of TPM1 was associated with depth of invasion and tumor node metastasis (TNM; p = 0.0030 and 0.0175, respectively). Furthermore, TPM1 might be a novel predictive biomarker for gastric cancer with an area under curve (AUC) of 0.8327. Overall survival analysis indicated that low TPM1 expression predicted poor survival (log-rank test, p = 0.0058)., Conclusions: TPM1 might be a novel predictive diagnostic and prognostic biomarker for gastric cancer (95% con-fidence interval = 0.7705 - 0.8949, p < 0.0001).

BACKGROUND The downregulation of tropomyosin 1 (TPM1) has been observed in various tumors, but few studies have focused on the clinical significance of TPM1 in intrahepatic cholangiocarcinoma (ICC). In the present study, we investigated the prognostic significance of TPM1 in ICC. MATERIAL AND METHODS A total of 124 patients with ICC were enrolled in this study. Quantitative real-time polymerase chain reaction (qRT-RCR) was performed to examine the mRNA levels of TPM1 in ICC tissue samples and adjacent noncancerous tissue specimens, while the protein level of TPM1 in tissue specimens were investigated using immunohistochemistry assay. The correlation of TPM1 with clinicopathological features of ICC was analyzed by chi-square test. Survival analysis was performed with Kaplan-Meier method. The Cox proportional hazards model was used to evaluate the prognostic value of TPM1 in patients with ICC. RESULTS TPM1 expression was significantly downregulated in ICC tissues at mRNA and protein levels (P<0.001 for both). Downregulated TPM1 mRNA was negatively associated with tumor size (P=0.001) and TNM stage (P=0.007). Moreover, survival analysis demonstrated that patients with low TPM1 expression had a shorter overall survival (OS) (P<0.001) and recurrence-free survival (RFS) (P<0.001) than those with high TPM1 expression. Additionally, multivariate analysis showed that TPM1 could be a potential biomarker for predicting the recurrence (HR=4.632, 95% CI: 3.832-10.368, P<0.001) and survival outcome (HR=5.320, 95% CI: 2.627-11.776, P<0.001) of ICC. CONCLUSIONS TPM1 may serve as a useful biomarker for predicting tumor recurrence and prognosis in patients with ICC.

Tropomyosin 4 (TPM4) has been found to be dys-regulated, and function as oncogene or anti-oncogene in human cancers. However, there was no report on the clinical significance and biological function of TPM4 in colon cancer. This study was designed to investigate the clinical value and biological function of TPM4 in colon cancer. Thus, we detected the TPM4 expression in colon cancer clinical samples, and conducted the gain-of-function in colon cancer cell lines. In our results, TPM4 mRNA and protein expressions were reduced in colon cancer tissues and cell lines compared with normal colon tissues and colon epithelial cell line, respectively. TPM4 protein low-expression was obviously associated with clinical stage, T classification (invasion depth), N classification (lymph node metastasis), distant metastasis and differentiation. Survival analysis showed low-expression of TPM4 was an unfavorable independent prognostic factor for colon cancer patients. Moreover, the experiments in vitro suggested up-regulated TPM4 expression suppressed colon cancer cell migration, invasion and metastasis-associated gene expression including MMP-2, MMP-9 and MT1-MMP, but had no effect on cell proliferation. In conclusion, TPM4 is associated with clinical progression in colon cancer patient and acts as a tumor suppressor in colon cancer cell., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

It is widely accepted that oral squamous cell carcinoma (OSCC) is a major contributor to the incidence and mortality of neck and head cancer. Tropomyosin-1 (TPM1), which is expressed at a low level, has been considered a prominent tumor-suppressing gene in a variety of solid tumors, although the precise mechanism of the TPM1 gene in OSCC progression remains unknown. We found that TPM1 expression levels decreased in OSCC patients and OSCC cell lines. The overall and cancer-specific survival of patients who exhibited low TPM1 levels were inferior to those of patients who had high TPM1 levels. It was also found that OSCC patients who suffered from disease stageⅠ-Ⅱ were more likely to have an up-regulated TPM1 expression level, and OSCC patients with lymph node metastasis had a higher probability of exhibiting reduced TPM1 expression. We show that overexpression of TPM1 can promote cell apoptosis and inhibit migration. Our results suggest that TPM1 can suppress tumors in OSCC, and the TPM1 expression level is related to OSCC patient prognosis., Competing Interests: The authors have declared that no competing interests exist.

We herein analyzed the relationships between tropomyosin protein expression levels and clinicopathological factors in order to determine the significance of tropomyosins in lung cancers. Although neoplastic cells expressed different isoforms of tropomyosin, overall expression levels were lower than those in bronchial and alveolar epithelial cells. In adenocarcinomas, tropomyosin levels were markedly reduced in poorly differentiated or solid subtype carcinomas, suggesting that a loss in the expression of tropomyosins is involved in the progression of lung adenocarcinomas. The potential utility of the immunohistochemical expression of tropomyosins for a histopathological diagnosis was also investigated. The sensitivity and specificity of a loss in the expression of tropomyosins were 100% and 50%, respectively, which were superior to those for the strong expression of p53 (sensitivity 100% and specificity 44%), a conventional biomarker. An immunohistochemical examination of tropomyosins may assist in the histopathological detection of lung cancer cells in small biopsy specimens.

In humans, four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) are known to produce a multitude of isoforms via alternate splicing and/or using alternate promoters. Expression of tropomyosin has been shown to be modulated at both the transcription and the translational levels. Tropomyosins are known to make up some of the stress fibers of human epithelial cells and differences in their expression has been demonstrated in malignant breast epithelial cell lines compared to 'normal' breast cell lines. We have recently reported the expression of four novel TPM1 isoforms (TPM1λ, TPM1µ, TPM1ν, and TPM1ξ) from human malignant tumor breast cell lines that are not expressed in adult and fetal cardiac tissue. Also, we evaluated their expression in relation to the stress fiber formation. In this study, nine malignant breast epithelial cell lines and three 'normal' breast cell lines were examined for stress fiber formation and expression of tropomyosin 2 (TPM2) isoform-specific RNAs and proteins. Stress fiber formation was assessed by immunofluorescence using Leica AF6000 Deconvolution microscope. Stress fiber formation was strong (++++) in the 'normal' cell lines and varied among the malignant cell lines (negative to +++). No new TPM2 gene RNA isoforms were identified, and TPM2β was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2β RNA or protein expression at high, statistically significant degrees. Previously, we had shown that TPM1δ and TPM1λ positively and inversely, respectively, correlated with stress fiber formation. The most powerful predictor of stress fiber formation was the combination of TPM2β RNA, TPM1δ RNA, and the inverse of TPM1λ RNA expression. Our results suggest that the increased expression of TPM1λ and the decreased expression of TPM1δ RNA and TPM2β may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells.

Polycystic ovary syndrome (PCOS) is a heterogenetic disorder in women that is characterized by arrested follicular growth and anovulatory infertility. The altered protein expression levels in the ovarian tissues reflect the molecular defects in folliculogenesis. To identify aberrant protein expression in PCOS, we analyzed protein expression profiles in the ovarian tissues of patients with PCOS. We identified a total of 18 protein spots that were differentially expressed in PCOS compared with healthy ovarian samples. A total of 13 proteins were upregulated and 5 proteins were downregulated. The expression levels of heat shock protein 90B1 (HSP90B1) and calcium signaling activator calmodulin 1 (CALM1) were increased by at least two-fold. The expression levels of HSP90B1 and CALM1 were positively associated with ovarian cell survival and negatively associated with caspase-3 activation and apoptosis. Knock-down of HSP90B1 with siRNA attenuated ovarian cell survival and increased apoptosis. In contrast, ovarian cell survival was improved and cell apoptosis was decreased in cells over-expressing HSP90B1. These results demonstrated the pivotal role of HSP90B1 in the proliferation and survival of ovarian cells, suggesting a critical role for HSP90B1 in the pathogenesis of PCOS. We also observed a downregulation of anti-inflammatory activity-related annexin A6 (ANXA6) and tropomyosin 2 (TPM2) compared with the normal controls, which could affect cell division and folliculogenesis in PCOS. This is the first study to identify novel altered gene expression in the ovarian tissues of patients with PCOS. These findings may have significant implications for future diagnostic and treatment strategies for PCOS using molecular interventions.

Jaw-closing muscles of several vertebrate species, including members of Carnivora, express a unique, "masticatory", isoform of myosin heavy chain, along with isoforms of other myofibrillar proteins that are not expressed in most other muscles. It is generally believed that the complement of myofibrillar isoforms in these muscles serves high force generation for capturing live prey, breaking down tough plant material and defensive biting. A unique isoform of tropomyosin (Tpm) was reported to be expressed in cat jaw-closing muscle, based upon two-dimensional gel mobility, peptide mapping, and immunohistochemistry. The objective of this study was to obtain protein and gene sequence information for this unique Tpm isoform. Samples of masseter (a jaw-closing muscle), tibialis (predominantly fast-twitch fibers), and the deep lateral gastrocnemius (predominantly slow-twitch fibers) were obtained from adult dogs. Expressed Tpm isoforms were cloned and sequencing yielded cDNAs that were identical to genomic predicted striated muscle Tpm1.1St(a,b,b,a) (historically referred to as αTpm), Tpm2.2St(a,b,b,a) (βTpm) and Tpm3.12St(a,b,b,a) (γTpm) isoforms (nomenclature reflects predominant tissue expression ("St"-striated muscle) and exon splicing pattern), as well as a novel 284 amino acid isoform observed in jaw-closing muscle that is identical to a genomic predicted product of the Tpm4 gene (δTpm) family. The novel isoform is designated as Tpm4.3St(a,b,b,a). The myofibrillar Tpm isoform expressed in dog masseter exhibits a unique electrophoretic mobility on gels containing 6 M urea, compared to other skeletal Tpm isoforms. To validate that the cloned Tpm4.3 isoform is the Tpm expressed in dog masseter, E. coli-expressed Tpm4.3 was electrophoresed in the presence of urea. Results demonstrate that Tpm4.3 has identical electrophoretic mobility to the unique dog masseter Tpm isoform and is of different mobility from that of muscle Tpm1.1, Tpm2.2 and Tpm3.12 isoforms. We conclude that the unique Tpm isoform in dog masseter is a product of the Tpm4 gene and that the 284 amino acid protein product of this gene represents a novel myofibrillar Tpm isoform never before observed to be expressed in striated muscle.

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and has been described as one of the deadliest of cancers affecting the genitourinary tract. Tropomyosin is a two-stranded α-helical coiled coil protein found in cell cytoskeletons. One of its isoforms, tropomyosin-1 (TPM1) has been reported as a novel tumor-suppressor gene and is downregulated in many solid tumors. However the expression level and function of TPM1 in RCC have not yet been determined. In the present study, we evaluated the TPM1-4 mRNA and TPM1 protein levels in RCC tissue samples. TPM1-overexpressing OSRC-2 and 786-O cell lines were also used to investigate the impact of TPM1 on RCC cells. We found that TPM1 was significantly and specifically downregulated in the RCC tissues. TPM1 expression was associated with tumor size, smoking status, Fuhrman grade and the prognosis of RCC patients. After TPM1 transfection, the migratory and invasive abilities of the OSRC-2 and 786-O cell lines were both reduced when compared to the control groups. Meanwhile, apoptosis was also enhanced in these two RCC cell lines following TPM1 transfection. Taken together, TPM1 exhibits characteristics of a tumor-suppressor gene while being overexpressed in RCC cell lines.

Heart disease is the number one killer in the USA, making cardiogenesis and its related pathways a relevant area of study for improving health and life expectancy. The Mexican salamander (axolotl), Ambystoma mexicanum, provides an excellent vertebrate animal model for studying myofibrillogenesis due to its naturally occurring cardiac nonfunction mutation. Homozygous recessive embryos do not develop normal hearts due to a lack of myofibril formation. In previous studies, myofibril-inducing ribonucleic acid (MIR) from the normal wild-type axolotl genome was found to rescue mutant nonfunctioning hearts through restoration of tropomyosin levels followed by normal myofibril formation. Our purpose in this study is to identify and characterize functional homologs for the MIR from human fetal heart ribonucleic acid (RNA). After randomized cloning of human fetal heart RNA, 396 clones were analyzed for rescuing ability by using mutant heart rescue bioassays and confocal microscopy. By these analyses, we discovered a functional homolog of MIR from human fetal heart RNA, which is associated with the mitochondrial cytochrome c oxidase subunit II gene. This RNA came from our clone #30 and induces tropomyosin synthesis and myofibrillogenesis in mutant axolotl hearts which ordinarily do not synthesize tropomyosin or form organized myofibrils. Clone #30, a mitochondrial RNA molecule associated with human cytochrome c oxidase, serves as a functional homolog of MIR, leading to tropomyosin production, organized myofibrils, and beating cardiac tissue in mutant hearts. These findings hold great potential for the treatment and repair of damaged hearts in patients who have suffered from myocardial infarctions and other heart diseases.

Food allergies are increasing worldwide, demonstrating a considerable public health concern. Shellfish allergy is one of the major food groups causing allergic sensitization among adults and children, affecting up to 2% of the general world population. Tropomyosin (TM) is the major allergen in shellfish and frequently used in the diagnosis of allergic sensitization and the detection of cross-contaminated food. To improve and establish better and more sensitive diagnostics for allergies and immunotherapeutics, large quantities of pure allergens are required. To establish a reproducible method for the generation of pure recombinant tropomyosin we utilized in this study different Escherichia coli strains (NM522, TOP10 and BL21(DE3)RIPL). In addition, isopropyl-β-D-thiogalactoside (IPTG) induction was compared with a novel auto-induction system to allow the generation of larger quantities of recombinant allergen. We demonstrated that the B-strain of E. coli is better for the expression of TM compared to the K-strain. Moreover, a higher yield could be achieved when using the auto-induction system, with up to 62 mg/l. High yield expressed recombinant TM from King prawn (KP) was compared to recombinant TM from Black tiger prawn (Pen m 1). We demonstrated that recombinant TM from KP and known isoallergen Pen m 1 have very similar molecular and immunological characteristics. Overall, we demonstrate that auto-induction can be used to express larger quantities of recombinant allergens for the development of diagnostic, to quantify allergens as well as immunotherapeutics employing isoallergens., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This paper reports that Slow-tonic S2 tropomyosin (Sst) and Slow tropomyosin isoform (Sti) was highly expressed in androgenic gland transcriptome of Macrobrachium nipponense, which may play crucial roles in sexual differentiation to maleness. In this study, two Sst and Sti gene homologues designated as Mnsst and Mnsti were cloned and characterized from a freshwater prawn M. nipponense. The full-length cDNA of Mnsst and Mnsti consists of 997 bp and 1926 bp, respectively, with an ORF of 852 bp encoding 284 amino acids, and the similarity in ORF reached to 95.82%. The deduced amino acid sequences of Mnsst and Mnsti shared the highest identity with Slow-tonic S2 tropomyosin and Slow tropomyosin isoform of Homarus americanus. Real-time quantitative RT-PCR showed that the Mnsst and Mnsti genes were expressed in different tissues with the highest level of expression in the androgenic gland, implying that these two genes may be related to sex-determination in M. nipponense. Real-time quantitative RT-PCR revealed that in addition, Mnsst and Mnsti were speculated to be related with embryonic organogenesis of M. nipponense, especially for the formation of complete mouthpart and digestive organ and stimulating larval changes of morphology and initiate metamorphosis, the results of present study implied that the two genes may play complex and important roles in sex differentiation of M. nipponense. Thus, we isolated two candidate genes that may advance the studies of sex-determination mechanism in M. nipponense and even the whole crustacean species, as well as promoting the all-male population culture of M. nipponense., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Neural crest cells are multipotent cells that migrate extensively throughout vertebrate embryos to form diverse lineages. Cell migration requires polarized, organized actin networks that provide the driving force for motility. Actin-binding proteins that regulate neural crest cell migration are just beginning to be defined., Results: We recently identified a number of actin-associated factors through proteomic profiling of methylated proteins in migratory neural crest cells. Here, we report the previously undocumented expression pattern of three of these proteins in chick early neural crest development: doublecortin (DCX), tropomyosin-1 (TPM-1), and actin depolymerizing factor (ADF). All three genes are expressed with varying degrees of specificity and intensity in premigratory and migratory neural crest cells, and their resulting proteins exhibit distinct subcellular localization in migratory neural crest cells. Morpholino knock down of ADF reveals it is required for Sox10 gene expression, but minimally important during neural crest migration., Conclusions: Neural crest cells express DCX, TPM-1, and ADF. ADF is necessary during neural crest specification, but largely dispensable for migration., (Copyright © 2013 Wiley Periodicals, Inc.)

Recent research has demonstrated that tropomyosin‑related kinase B (TrkB) plays an important role in neuronal survival, differentiation and migration; yet, its specific role in human nasopharyngeal carcinoma (NPC) is unclear. To elucidate its role in NPC, we examined TrkB expression in NPC tissues and cell lines, and investigated the correlation between TrkB expression and prognosis in patients with NPC. Immunohistochemical analysis was performed on NPC specimens from 108 patients with follow-up information. Additionally, reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analyses were used to determine TrkB expression levels in NPC and noncancerous nasopharyngeal tissues. RT-PCR and western blot analyses were also used to determine TrkB expression levels in 7 NPC cell lines and a nasopharyngeal epithelium cell line. High TrkB expression was significantly correlated with T classification, lymph node metastasis and clinical stage, respectively. Patients with NPC who had high TrkB expression had reduced disease-free survival and overall survival when compared with patients who had low TrkB expression. Multivariate Cox regression analysis revealed that TrkB overexpression was an independent prognostic factor for patients with NPC. Furthermore, TrkB was overexpressed in NPC specimens and cell lines. TrkB expression levels were significantly increased in NPC specimens, and enhanced levels were correlated with a poor prognosis in patients with NPC. These findings suggest that TrkB may contribute to NPC progression and represent a novel prognostic indicator for patients with NPC.

Thin filament-associated proteins such as calponin, caldesmon, tropomyosin, and smoothelin are thought to regulate acto-myosin interaction and thus, muscle contraction. However, the effect of inflammation on the expression of thin filament-associated proteins is not known. The aim of the present study is to determine the changes in the expression of calponin, caldesmon, tropomyosin, and smoothelin in colonic smooth muscle from trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulphate (DSS)-induced colitis in mice. Expression of h-caldesmon, h2-calponin, α-tropomyosin, and smoothelin-A was measured by qRT-PCR and Western blot. Contraction in response to acetylcholine in dispersed muscle cells was measured by scanning micrometry. mRNA and protein expression of α-actin, h2-calponin, h-caldesmon, smoothelin, and α-tropomyosin in colonic muscle strips from mice with TNBS- or DSS-induced colitis was significantly increased compared to control animals. Contraction in response to acetylcholine was significantly decreased in muscle cells isolated from inflamed regions of TNBS- or DSS-treated mice compared to control mice. Our results show that increase in the expression of thin filament-associated contractile proteins, which inhibit acto-myosin interaction, could contribute to decrease in smooth muscle contraction in inflammation.

Atherosclerosis is a chronic inflammatory disorder that occurs as a result of mononuclear lymphocyte infiltration to the arterial wall, smooth muscle cell proliferation and damage in the arterial wall caused by extracellular matrix accumulation. Besides several genetic and environmental factors, increased serum cholesterol and oxidized low density lipoproteins are considered to be major inducing factors of atherosclerosis. Several protective agents have been used to prevent the progression of atherosclerosis and recently vitamin E has been focused because of its significant role in signaling mechanisms. Since many different cell types are involved in the development of hypercholesterolemia induced atherosclerosis, it is important to investigate wide range of proteins to highlight the pathologic and diagnostic mechanisms. In this study, by using proteomic technique, we identified differentially expressed proteins following cholesterol and also vitamin E treatments. The expressions of apolipoprotein A I and apolipoprotein E involved in lipid metabolism, peroxiredoxin 1, peroxiredoxin 2 and thioredoxin involved in antioxidant system, 14-3-3 protein zeta delta and 14-3-3 protein beta alpha in cell signaling, biglycan, vimentin, tropomyosin and smooth muscle α-actin as structural and contractile proteins have been discussed., Biological Significance: We observed several protein alterations in aorta of cholesterol fed and vitamin E treated rabbits.These differentially expressed proteins associated with key mechanisms involved in atherosclerosis and signaling mechanisms related with vitamin E. These findings for different proteins might be helpful for deciphering the pathogenesis in atherosclerosis. In addition it provides a new perspective to understand mechanisms of beneficial effect of vitamin E on the signaling pathways in atherogenesis. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We determined the isoforms of tropomyosin expressed and the level of tropomyosin phosphorylation in donor, end-stage failing and hypertrophic obstructive cardiomyopathy samples of human heart muscle. Western blots and isoform-specific antibodies showed that α-tropomyosin was the only significant isoform expressed and that tropomyosin was 25-30% phosphorylated at serine 283. Mass spectrometry confirmed directly that α-tropomyosin made up over 95% of tropomyosin but also indicated the presence of up to 4% κ-tropomyosin and much smaller amounts of β-, γ- and smooth β-tropomyosin and about 26% phosphorylation. Neither the isoform distribution nor the level of phosphorylation changed significantly in the pathological heart muscle samples.

We reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of dog extraocular muscles. Many dog extraocular fibers, especially orbital fibers, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. Additional observations suggested possible differences in the tropomyosin (Tm) and troponin T (TnT) isoform composition of global and orbital fibers. Therefore, we tested, using SDS-PAGE and immunoblotting, whether differences in Tm and TnT isoform expression do, in fact, exist between global and orbital layers of dog and rat EOMs and to compare expression patterns among identified fast and slow single fibers from both muscle layers. The Tm isoforms expressed in global fast and slow fibers are the same as in limb fast (α-Tm and β-Tm) and slow (γ-Tm and β-Tm) fibers, respectively. Orbital slow orbital fibers, on the other hand, each co-express all three sarcomeric Tm isoforms (α, β and γ). The results indicate that fast global and orbital fibers express only fast isoforms of TnT, but the relative amounts of the individual isoforms are different from those in limb fast muscle fibers and an abundant fast TnT isoform in the orbital layer was not detected in fast limb muscles. Slow fibers in both layers express slow TnT isoforms and the relative amounts also differ from those in limb slow fibers. Unexpectedly, significant amounts of cardiac TnT isoforms were also detected in slow fibers, especially in the orbital layer in both species. TnI and TnC isoform patterns are the same as in fast and slow fibers in limb muscles. These results expand the understanding of the elaborate diversity in contractile protein isoform expression in mammalian extraocular muscle fibers and suggest that major differences in calcium-activation properties exist among these fibers, based upon Tm and TnT isoform expression patterns.

Objective: To identify microRNAs (miRNAs) that are overexpressed in renal cell carcinoma (RCC) and characterize the functional role of miR-21., Materials and Methods: The miRNA expression profiles between RCC tissue and adjacent normal tissue were compared using microarray analysis. The differential expression of miR-21 was validated by real-time polymerase chain reaction (RT-PCR). 786-O RCC cells were transfected with miR-21 mimic, miR-21 inhibitor, or negative controls and cell proliferation, apoptosis and cell cycle were examined by MTT assay and flow cytometry. The expression of programmed cell death 4 (PDCD4) and tropomyosin 1 (TPM1) was detected by RT-PCR and Western blot analysis., Results: Compared to adjacent normal tissue, 10 human miRNAs were significantly upregulated and 7 were downregulated in RCC tissue. RT-PCR confirmed that miR-21 was significantly overexpressed in RCC tissue. In vitro expression of miR-21 mimic promoted the growth of 786-O cells, whereas miR-21 inhibitor inhibited cell growth by inducing apoptosis and cell cycle arrest at S phase. Furthermore, miR-21 mimic or inhibitor significantly reduced or increased the expression of PDCD4 and TPM1., Conclusions: MiR-21 is overexpressed in RCC tissue and modulates the growth, apoptosis and cell cycle progression of RCC cells and regulates the expression of PDCD4 and TPM1.

α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca(2+) sensitivity and cardiomyocyte contractility without changes in intracellular Ca(2+) transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative gene regulators. Alterations in the expression of miRNAs have been implicated in the pathogenesis and development of most human malignancies. Recent data indicate that microRNA-21 and microRNA-10b are significantly elevated in glioblastoma multiforme (GBM) suggesting their role in the regulation of multiple genes associated with cancer. In this study, U87MG human glioblastoma cells were treated with miRNA inhibitors targeting miR-10b and miR-21, alone or in combination. The results showed that the miR-21 inhibitor additively interacted with miR-10b inhibitor on U87MG cells. The 50% inhibitory concentration values were dramatically decreased in cells treated with the combination of miR-10b and miR-21 inhibitors. Furthermore, inhibitors synergistically combined, enhanced apoptosis significantly and reduced invasion ability assessed by flow cytometry and Transwell migration assay. Thus, the miR-21 inhibitor may interrupt the activity of EGFR pathways, increasing PDCD4 and TPM1 expression and reducing MMP activities, independently of PTEN status. Meanwhile, miR-10b inhibitor reduced by Twist proceeds to inhibit translation of the mRNA encoding HOXD10 leading to the increase of the expression of the well-characterized pro-metastatic gene RHOC. Taken together, these data strongly suggest that a combination of miR-21 inhibitor and miR-10b inhibitor could be an effective therapeutic strategy for controlling the growth of GBM by inhibiting oncogene expression and overexpressing tumor suppressor genes. Moreover, a regulatory strategy based on the combination of miRNA inhibitors may provide insights into the mechanisms of the modulation of signaling genes involved in tumor cell apoptosis and invasiveness.

Objective: To investigate the role of inflammation in the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy (HCM)., Design: Clinical study., Setting: Kuopio University Hospital and University of Eastern Finland, Kuopio, Finland., Subjects: Twenty-four patients with a single HCM-causing mutation D175N in the α-tropomyosin gene and 17 control subjects., Main Outcome Measures: Endomyocardial biopsy samples taken from the patients with HCM were compared with matched myocardial autopsy specimens. Levels of high-sensitivity C-reactive protein (hsCRP) and proinflammatory cytokines were measured in patients and controls. Myocardial late gadolinium enhancement (LGE) in cardiac MRI (CMRI) was detected., Results: Endomyocardial samples in patients with HCM showed variable myocyte hypertrophy and size heterogeneity, myofibre disarray, fibrosis, inflammatory cell infiltration and nuclear factor kappa B (NF-κB) activation. Levels of hsCRP and interleukins (IL-1β, IL-1RA, IL-6, IL-10) were significantly higher in patients with HCM than in control subjects. In patients with HCM, there was a significant association between the degree of myocardial inflammatory cell infiltration, fibrosis in histopathological samples and myocardial LGE in CMRI. Levels of hsCRP were significantly associated with histopathological myocardial fibrosis. hsCRP, tumour necrosis factor α and IL-1RA levels had significant correlations with LGE in CMRI., Conclusions: A variable myocardial and systemic inflammatory response was demonstrated in patients with HCM attributable to an identified sarcometric mutation. Inflammatory response was associated with myocardial fibrosis, suggesting that myocardial fibrosis in HCM is an active process modified by an inflammatory response.

Cucujus clavipes puniceus (C.c.p.) is a nonmodel, freeze-avoiding beetle that overwinters as extremely cold-tolerant larvae in the interior boreal forests of Alaska to temperatures as low as -100 °C. Using a tandem MS-based approach, we compared the proteomes of winter- and summer-collected C.c.p. to identify proteins that may play functional roles in successful overwintering. Using Gene Ontology (GO) analysis and manual interpretation, we identified 104 proteins in winter and 128 proteins in summer samples. We found evidence to indicate a cytoskeletal rearrangement between seasons, with Winter NDSC possessing unique actin and myosin isoforms while summer larvae up-regulated α actinin, tubulin, and tropomyosin. We also detected a fortification of the cuticle in winter via unique cuticle proteins, specifically larval/pupal rigid cuticle protein 66 precursor and larval cuticle protein A2B. Also, of particular interest in the winter larvae was an up-regulation of proteins related to silencing of genes (bromodomain adjacent to zinc finger domain 2A and antisilencing protein 1), proteins involved with metabolism of amines (2-isopropylmalate synthase and dihydrofolate reductase), and immune system process (lysozyme C precursor), among others. This represents the first high throughput MS/MS analysis of a nonmodel, cold-tolerant organism without a concurrent microarray analysis.

Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We previously showed that complete loss of smooth muscle myosin heavy chain isoform 2 (SM2) resulted in postnatal lethality, but in het mice a partial loss of SM2 (SM2(+/-)) was accompanied by down-regulation of SM1 with unaltered SM2:SM1 ratio. To determine whether a normal bladder function would be maintained throughout its lifespan, we aged WT and SM2(+/-) mice up to 18 months and analyzed a) SM2:SM1 ratio b) bladder smooth muscle structure and c) function in SM2(+/-) het mice. A notable finding was that ~50% of 15-18 months old male SM2(+/-) mice exhibited urinary retention in bladder with the distention of upper urethra. In SM2(+/-) mouse bladder with urinary retention, the SM2:SM1 ratio was decreased but not in SM2(+/-) mouse bladder that did not develop urinary retention. Interestingly in the distended bladder the expression levels of α-actin and tropomyosin remained unaltered despite a reduction in the number of myosin thick filaments. These distended bladders showed hypersensitivity to submaximal K(+) depolarization and M3-receptor stimulation, without a significant increase in myosin light chain phosphorylation. We therefore suggest that a partial loss of SM2 may predispose male mice to develop lower urinary tract obstruction during ageing. In addition our data suggest that bladder obstruction can cause a further reduction in SM2 expression and SM2:SM1 ratio, and a hyper-contractility of the bladder smooth muscle.

L-Arginine is a conditionally essential amino acid for humans and plays an important role in the regulation of cardiovascular function and antioxidative defense. Previous studies have focused on the important role of L-arginine as a physiological precursor in the generation of nitric oxide and polyamines in endothelial cells (cells that line the interior surface of blood vessels). Because of the rapid development of high-throughput proteomics technology, there is now growing interest in studying roles for L-arginine in modulating endothelial-cell protein expression. Of particular interest, recent proteomics analysis has shown that treatment of coronary venular endothelial cells with a physiological level of L-arginine (e.g., 0.1 mM) increases expression of structural proteins (vimentin and tropomyosin) and cytochrome bc1 complex iii-chain A, while decreasing expression of stress-related proteins (PDZ domain containing-3), in these cells. These findings aid in elucidating the mechanisms responsible for the beneficial effect of physiological levels of L-arginine on the circulatory system.

Background:  The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known., Methods: Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae., Key Results: Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae., Conclusions & Inferences: These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism.

Tropomyosin (TM) plays a central role in calcium mediated striated muscle contraction. There are three muscle TM isoforms: alpha-TM, beta-TM, and gamma-TM. alpha-TM is the predominant cardiac and skeletal muscle isoform. beta-TM is expressed in skeletal and embryonic cardiac muscle. gamma-TM is expressed in slow-twitch musculature, but is not found in the heart. Our previous work established that muscle TM isoforms confer different physiological properties to the cardiac sarcomere. To determine whether one of these isoforms is dominant in dictating its functional properties, we generated single and double transgenic mice expressing beta-TM and/or gamma-TM in the heart, in addition to the endogenously expressed alpha-TM. Results show significant TM protein expression in the betagamma-DTG hearts: alpha-TM: 36%, beta-TM: 32%, and gamma-TM: 32%. These betagamma-DTG mice do not develop pathological abnormalities; however, they exhibit a hyper contractile phenotype with decreased myofilament calcium sensitivity, similar to gamma-TM transgenic hearts. Biophysical studies indicate that gamma-TM is more rigid than either alpha-TM or beta-TM. This is the first report showing that with approximately equivalent levels of expression within the same tissue, there is a functional dominance of gamma-TM over alpha-TM or beta-TM in regulating physiological performance of the striated muscle sarcomere. In addition to the effect expression of gamma-TM has on Ca(2+) activation of the cardiac myofilaments, our data demonstrates an effect on cooperative activation of the thin filament by strongly bound rigor cross-bridges. This is significant in relation to current ideas on the control mechanism of the steep relation between Ca(2+) and tension.

Background: Since hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, it is still important to understand hepatocarcinogenesis mechanisms and identify effective markers for tumor progression to improve prognosis. Amplification and overexpression of Tropomyosin3 (TPM3) are frequently observed in HCC, but its biological meanings have not been properly defined. In this study, we aimed to elucidate the roles of TPM3 and related molecular mechanisms., Methods: TPM3-siRNA was transfected into 2 HCC cell lines, HepG2 and SNU-475, which had shown overexpression of TPM3. Knockdown of TPM3 was verified by real-time qRT-PCR and western blotting targeting TPM3. Migration and invasion potentials were examined using transwell membrane assays. Cell growth capacity was examined by colony formation and soft agar assays., Results: Silencing TPM3 resulted in significant suppression of migration and invasion capacities in both HCC cell lines. To elucidate the mechanisms behind suppressed migration and invasiveness, we examined expression levels of Snail and E-cadherin known to be related to epithelial-mesenchymal transition (EMT) after TPM3 knockdown. In the TPM3 knockdown cells, E-cadherin expression was significantly upregulated and Snail downregulated compared with negative control. TPM3 knockdown also inhibited colony formation and anchorage independent growth of HCC cells., Conclusions: Based on our findings, we formulate a hypothesis that overexpression of TPM3 activates Snail mediated EMT, which will repress E-cadherin expression and that it confers migration or invasion potentials to HCC cells during hepatocarcinogenesis. To our knowledge, this is the first evidence that TPM3 gets involved in migration and invasion of HCCs by modifying EMT pathway.

The ubiquitin-proteasome system plays an important role in the degradation of myofibrillar proteins that occurs in muscle wasting. Many studies have demonstrated the importance of enzymes mediating conjugation of ubiquitin. However, little is known about the role of deubiquitinating enzymes. We previously showed that the USP19-deubiquitinating enzyme is induced in atrophying skeletal muscle (Combaret L, Adegoke OA, Bedard N, Baracos V, Attaix D, Wing SS. Am J Physiol Endocrinol Metab 288: E693-E700, 2005). To further explore the role of USP19, we used small interfering RNA (siRNA) in L6 muscle cells. Lowering USP19 by 70-90% in myotubes resulted in a 20% decrease in the rate of proteolysis and an 18% decrease in the rate of protein synthesis, with no net change in protein content. Despite the decrease in overall synthesis, there were approximately 1.5-fold increases in protein levels of myosin heavy chain (MHC), actin, and troponin T and a approximately 2.5-fold increase in tropomyosin. USP19 depletion also increased MHC and tropomyosin mRNA levels, suggesting that this effect is due to increased transcription. Consistent with this, USP19 depletion increased myogenin protein and mRNA levels approximately twofold. Lowering myogenin using siRNA prevented the increase in MHC and tropomyosin upon USP19 depletion, indicating that myogenin mediated the increase in myofibrillar proteins. Dexamethasone treatment lowered MHC and increased USP19. Depletion of USP19 reversed the dexamethasone suppression of MHC. These studies demonstrate that USP19 modulates transcription of major myofibrillar proteins and indicate that the ubiquitin system not only mediates the increased protein breakdown but is also involved in the decreased protein synthesis in atrophying skeletal muscle.

The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c) embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR), is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c) axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM) expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR) are similar to untreated normal. However, at the protein level, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.

Myofibrillar protein tropomyosin (TM) is a normal physiological protein participating in regulation of muscular contraction. It is widely prevalent among living organisms. This explains cross-reactivity of allergic patients to home dust, sea fish, cockroaches, etc. The presence of similar IgE-binding epitopes in TM of different origin is a key factor in development of cross-reactivity (CR). CR to TM is a general biological phenomenon. We consider modified TM as a basic component in design of allergovaccines of a new generation.

Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection.

To analyze the potential of RNA interference (RNAi) in the intestinal nematode Heligmosomoides polygyrus, we delivered double-stranded RNA (dsRNA) of tropomyosin to various life stages of the parasite. Three different methods were examined for their potential use. First, feeding of recombinant bacteria that expressed dsRNA did neither result in phenotypical changes of H. polygyrus nor in a significant reduction of tropomyosin mRNA levels. In contrast, feeding of such bacteria to Caenorhabditis elegans elicited the expected phenotypes. Quantification of bacteria ingested by C. elegans and H. polygyrus larvae (L1) revealed that the parasitic worms took up only a fraction of the bacteria ingested by C. elegans. Second, electroporation of L1 failed to transport siRNA through the cuticle and was lethal to the larvae. However, the cuticle of adult worms was penetrated by dye-labeled RNA, but no systemic spreading was observed. Third, soaking of adult H. polygyrus in tropomyosin dsRNA led to a higher proportion of worms showing symptoms of ageing, such as a disintegrated gut and ovaries, but did not induce reduction of tropomyosin mRNA levels. Database analysis revealed that orthologous proteins involved in dsRNA-uptake and -systemic spread in C. elegans are missing in the parasitic nematodes Brugia malayi and Haemonchus contortus, whereas proteins responsible for dsRNA-processing, -amplification and mRNA-regulation are present. Thus, our data indicate that the study of gene function by RNAi in H. polygyrus is limited, possibly due to deficiencies of genes involved in RNA-uptake and spread.

The creation of the contractile apparatus in muscle involves the co-activation of a group of genes encoding muscle-specific proteins and the production of high levels of protein in a short period of time. We have studied the transcriptional control of six Drosophila muscle genes that have similar expression profiles and we have compared these mechanisms with those employed to control the distinct expression profiles of other Drosophila genes. The regulatory elements controlling the transcription of co-expressed muscle genes share an Upstream Regulatory Element and an Intronic Regulatory Element. Moreover, similar clusters of MEF2 and CF2 binding sites are present in these elements. Here, we demonstrate that CF2 depletion alters the relative expression of thin and thick filament components. We propose that the appropriate rapid gene expression responses during muscle formation and the maintenance of each muscle type is guaranteed in Drosophila by equivalent duplicate enhancer-like elements. This mechanism may be exceptional and restricted to muscle genes, reflecting the specific requirement to mediate rapid muscle responses. However, it may also be a more general mechanism to control the correct levels of gene expression during development in each cell type.

Background and Purpose: Tropomyosin (TM) is an actin-binding protein, which is localized head to tail along the length of the actin filament. There are three major TM isoforms in human striated muscle. Mutations in beta-tropomyosin (TPM2) have recently been identified as an important cause of neuromuscular disorders., Materials and Methods: The expression of TM isoforms in patients carrying mutations in TPM2 was detected using a combination of SDS-PAGE, Western blotting, and a new method to measure the relative abundance of the various TM transcripts., Results: The level of gamma-TM is reduced in patients with mutations in TPM2. Beta-tropomyosin was expressed at high levels in muscle specimens of the patients., Discussion: Our study indicates that beta-TM gene mutations can alter the expression of other sarcomeric TM isoforms and that the perturbation of TM isoform levels may affect the dimer preference within the thin filaments, which may contribute to muscle weakness as a result of both functional and structural changes in muscle.

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level. Our previous studies suggest that mir-21 functions as an oncogene and has a role in tumorigenesis, in part through regulation of the tumor suppressor gene tropomyosin 1 (TPM1). Given that TPM1 has been implicated in cell migration, in this study we further investigated the role of mir-21 in cell invasion and tumor metastasis. We found that suppression of mir-21 in metastatic breast cancer MDA-MB-231 cells significantly reduced invasion and lung metastasis. Consistent with this, ectopic expression of TPM1 remarkably reduced cell invasion. Furthermore, we identified two additional direct mir-21 targets, programmed cell death 4 (PDCD4) and maspin, both of which have been implicated in invasion and metastasis. Like TPM1, PDCD4 and maspin also reduced invasiveness of MDA-MB-231 cells. Finally, the expression of PDCD4 and maspin inversely correlated with mir-21 expression in human breast tumor specimens, indicating the potential regulation of PDCD4 and maspin by mir-21 in these tumors. Taken together, the results suggest that, as an oncogenic miRNA, mir-21 has a role not only in tumor growth but also in invasion and tumor metastasis by targeting multiple tumor/metastasis suppressor genes. Therefore, suppression of mir-21 may provide a novel approach for the treatment of advanced cancers.

The evolution from unicellular to multicellular organisms of increasing complexity is paralleled by increased numbers of tropomyosin (Tm) genes and increasing numbers ofisoforms encoded by each gene. The regulation of Tm isoform expression is intimately associated with the morphological changes that take place during development and cell differentiation. The tissue- and cell-specific Tm expression patterns are regulated at multiple levels, allowing precise spatial and temporal regulation of Tm expression. In this chapter, we review the Tm isoform expression pattern during differentiation of different tissue types and from this data infer some general principles regarding Tm expression patterns during differentiation. Finally, we review the mechanisms that account for the highly regulated repertoire ofTm isoform expression.

Storage mites have been recognized as a cause of asthma and rhinitis. Studies from several countries have shown that the IgE-mediated allergy to storage mites is of considerable importance, especially in rural populations. This study aimed to identify and characterize new allergens from Tyrophagus putrescentiae. A partial cDNA sequence encoding tropomyosin was isolated from the cDNA library by immunoscreening using anti-mouse IgG1 sera raised against T. putrescentiae whole body extract. The deduced amino acid sequence shares 64-94% identity with previously known allergenic tropomyosins. Its recombinant protein was produced by using a pET 28b expression system and purified by affinity chromatography using Ni-NTA agarose. The IgE reactivities of tropomyosins from T. putrescentiae and Dermatophagoides farinae were compared by enzyme linked immunosorbent assay (ELISA). Recombinant Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity against the same sera from storage mite-sensitized and house dust mite-sensitized subjects. Both recombinant Tyr p 10 and Der f 10 showed little inhibition of IgE binding to T. putrescentiae crude extract by ELISA. Tropomyosin seems to contribute only a small portion of the cross-reactivity with house dust mites.

Blo t 11, a dust-mite (Blomia tropicalis) paramyosin, is an allergen with significant IgE reactivity that has potential as a diagnostic/therapeutic reagent for house-dust-mite allergy. The present study describes the successful expression of Blo t 11 and its immunodominant peptide fD in insect cells using a baculovirus expression system. The Blo t 11 and fD genes were cloned into the pMelBacA vector and the resulting vectors were co-transfected into Sf9 insect cells with Bac-N-Blue DNA. Plaque assay was used to select for recombinant virus that was then used to infect High Five insect cells for protein expression. Secreted proteins were harvested by immuno-affinity purification using monoclonal antibodies to Blo t 11. Purified proteins were analysed by immunoblotting, N-terminal sequencing and ELISA. Immunoblot analyses revealed the full-length Blo t 11 cDNA expressed as a minor protein band of approx. 200 kDa and two major protein bands of approx. 60 and 70 kDa. Clones expressing fD cDNA fragment produced a protein of approx. 30 kDa that was confirmed to be fD by N-terminal sequencing. Approx. 4-7.5 mg/l of fD and 1 mg/l of Blo t 11 were obtained by affinity purification. ELISA results showed that human IgE reactivity to these recombinant allergens was lower as compared with that of the native Blo t 11, suggesting that these baculovirus-expressed allergens exhibiting reduced allergenicity could be useful for the development of immunotherapeutic reagents for mite allergy.

The possible use of cytoskeletal components as biomarkers of organic pollution in mussels has been investigated. Responses of non-muscular actin and tropomyosin (TM), two bivalve proteins that were recently demonstrated to cross-react with anti-fish-CYP1A, were analysed in digestive tissue of blue mussels (Mytilus sp.) exposed to a wide range of organic contaminants. The results were evaluated with ELISA and Western blot assays, utilising commercial monoclonal antibodies, and compared with expression of Hsp70, a marker of chemical stress. Furthermore, mussels were sampled from the Baltic Sea at sites with different degrees of pollution to assess the expression of these proteins, and to monitor seasonal changes in relation to energy reserves and water temperature. The results demonstrated that expression of microsomal actin was significantly higher (p<0.02) in mussels exposed to a brominated flame retardant (BDE-47), and lower, however not significantly, in specimens exposed to crude oil, alone and spiked with alkylphenols and PAHs. Hsp70 was strongly induced in all exposure groups, which also included bisphenol A and diallylphthalate. Furthermore, microsomal actin exhibited seasonal variations, and expression was negatively correlated with water temperature. No correlation was seen between actin and the microfilament-binding protein TM, indicating that regulation of these two cytoskeletal components are not coupled. Furthermore, parallel and significant (p<0.05) up-regulations of TM and Hsp70 were seen in individuals sampled from a strongly polluted field site, whereas the seasonal analysis showed that TM expression was positively correlated with energy reserves (total glycogen content) in mussels, suggesting the use of TM as a marker of growth. In conclusion, this study has demonstrated the cytoskeleton to be a target of contaminants in mussels, calling for further attention. Exposure-induced increase of microsomal actin can be interpreted either as stimulated actin synthesis, or re-arrangements of the dynamic microfilaments.

Heterogeneity and tissue specificity of tropomyosin isoforms obtained from four species of bivalves (Scapharca broughtonii (ark shell), Mytilus galloprovincialis (mussel), Atrina pectinata (surf clam) and Crassostrea gigas (Pacific oyster)), were examined. Tropomyosins were extracted from translucent and opaque portions of posterior adductor muscle, respectively, and cardiac muscle of each bivalve. There were two tropomyosin isoforms in the ark shell, the surf clam and the Pacific oyster. They were designated as TMa and TMb. In the ark shell, TMa was the common isoform and TMb was specific for the opaque portion of the adductor muscle. In the surf clam, TMb was the common isoform present in all tissues. TMa was found only in the translucent portion of muscle. In the Pacific oyster, TMb was the major component in both portions of adductor muscle and TMa was the major component in cardiac muscle, although both tropomyosins were included in all tissues. The mussel had only one tropomyosin.

Aims: Obstruction of the urinary bladder outlet induces detrusor smooth muscle (DSM) hypertrophy. The goal of this study was to determine whether the composition of thin filament-associated proteins, known to play important roles in cytoskeletal structure and/or the regulation of contraction, is altered in DSM during hypertrophy., Methods: DSM hypertrophy was induced in male rabbits by partial ligation of the urethra. Sham-operated rabbits served as a control. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR revealed a significant increase in the expression of mRNAs for basic (h1) calponin (CaP), and alpha-isoform of tropomyosin (Tm) in hypertrophied DSM compared to controls. Western blotting and two-dimensional (2-D) gel electrophoresis showed enhanced expression of these proteins and also a significant increase in the expression of beta-non muscle and gamma-smooth muscle actin in the DSM from obstructed bladders, while alpha-actin remained constant., Results: Enhanced expression of these proteins in the DSM from obstructed bladders was confirmed by immunofluorescence microscopy. Double immunostaining with Cap/Tm and alpha/beta-actin-specific antibodies showed co-localization of these proteins in myocytes. Colocalization of smooth muscle specific myosin and CaP to cytoplasmic filaments in cells dissociated from the hypertrophied DSM indicated that these cells are differentiated smooth muscle cells., Conclusions: The change in the isoforms of actin, Cap, and Tm may be part of the molecular mechanism for bladder compensation in increased urethral resistance., (Neurourol. Urodynam. (c) 2005 Wiley-Liss, Inc.)

Neoplastic transformation by Ras proteins markedly suppresses the expression of certain isoforms of tropomyosins (TMs), which are important regulators of actin cytoskeleton. Downregulation of TMs and other actin-associated proteins is believed to result in the assembly of aberrant cytoskeleton, which in turn contributes to the malignant transformation by Ras. Oncogenic activation of ras, in addition to suppressing TMs by means of epigenetic mechanisms, also rapidly inhibits their cytoskeletal fractionation, leading to the disruption of cytoskeleton. Restoration of expression of certain isoforms of TMs reorganizes microfilaments and suppresses the malignant growth of ras-transformed cells. This chapter discusses some of the approaches to the analysis of TM isoform expression in normal and ras-transformed cells.

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H2O2 generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tgp22phox VSMC in an in vitro wound scratch model. Enhanced H2O2 production in Tgp22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H2O2 than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle alpha-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tgp22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased alpha-actin.

Purpose: The thin filament associated proteins caldesmon, tropomyosin and calponin have been shown to modulate actin-myosin interaction, actomyosin adenosine triphosphatase and contraction in smooth muscle. This study was performed to determine whether the expression of these proteins is altered in diabetes induced decrease in the contractility of bladder wall smooth muscle., Materials and Methods: Detrusor samples were obtained from New Zealand White male rabbits with alloxan induced diabetes, and from age and sex matched control rabbits. In addition, a bladder myocyte cell line, which continues to express smooth muscle phenotype, was exposed to either normal (5 mM) or high (50 mM) concentrations of glucose. The levels of expression of the thin filament associated proteins were determined at the mRNA and protein levels by reverse transcriptase-polymerase chain reaction and Western blotting, respectively., Results: Detrusor smooth muscle tissue from rabbits with alloxan induced diabetes showed over expression of thin filament associated proteins, calponin, tropomyosin and caldesmon when compared with that of the control. Similar up-regulation was seen also in bladder myocytes in cultures treated with 50 mM glucose, indicating that the high glucose induced the changes., Conclusions: Our results suggest that the increased expression of thin filament proteins, calponin, tropomyosin and caldesmon in diabetic rabbits might alter the contractile and cytoskeletal structure in bladder myocytes. The over expression of these thin filament associated proteins, which suppresses actin-myosin interaction and actomyosin adenosine triphosphatase, and the enhancement of this suppression by tropomyosin are likely to have an effect on the relationship between force and myosin light chain phosphorylation, requiring higher levels of phosphorylation in diabetic detrusor compared with that of control. The downstream effects of high glucose (eg oxidative stress) appear to modulate the transcriptional regulation of thin filament mediated regulatory proteins in bladder smooth muscle.