Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1µ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and µ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1µ non-muscle isoform protein expression before and during cardiac differentiation.

Tropomyosin Isoform Diversity in the Cynomolgus Monkey Heart and Skeletal Muscles Compared to Human Tissues.

Old world monkeys separated from the great apes, including the ancestor of humans, about 25 million years ago, but most of the genes in humans and various nonhuman primates are quite similar even though their anatomical appearances are quite different. Like other mammals, primates have four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing. Only TPM1 produces two sarcomeric isoforms (TPM1α and TPM1κ), and TPM2, TPM3, and TPM4 each generate one sarcomeric isoform. We have cloned and sequenced TPM1α, TPM1κ, TPM2α, TPM3α, and TPM4α with RNA from cynomolgus (Cyn) monkey hearts and skeletal muscle. We believe this is the first report of directly cloning and sequencing of these monkey transcripts. In the Cyn monkey heart, the rank order of TPM isoform expression is TPM1α > TPM2α > TPM1κ > TPM3α > TPM4α. In the Cyn monkey skeletal muscle, the rank order of expression is TPM1α > TPM2α > TPM3α > TPM1κ > TPM4α. The major differences in the human heart are the increased expression of TPM1κ, although TPM1α is still the dominant transcript. In the Cyn monkey heart, the only sarcomeric TPM isoform at the protein level is TPM1α. This is in contrast to human hearts where TPM1α is the major sarcomeric isoform but a lower quantity of TPM1κ, TPM2α, and TPM3α is also detected at the protein level. These differences of tropomyosin and/or other cardiac protein expression in human and Cyn monkey hearts may reflect the differences in physiological activities in daily life.

Tumor Lysis Syndrome in a Low-Risk Pancreatic Cancer Patient.

Tumor lysis syndrome (TLS) is the most common hematologic emergency encountered during the treatment of high-grade malignancies. While it can lead to death, the prognosis is typically excellent if caught early on in the course. Risk stratification prior to treatment initiation is paramount in deciding the utility of prophylaxis and ultimately in reducing morbidity and mortality. The following case describes the development of TLS in a patient categorized as low risk and highlights the need for further elucidation of a unified risk stratification system.

Effect of MG-132 on myofibrillogenesis and the ubiquitination of GAPDH in quail myotubes.

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.

Identification of a novel TPM4 isoform transcript and comparison to the expression of other tropomyosin isoforms in bovine cardiac and skeletal muscles.

In mammals, there are four tropomyosin (TPM) genes (TPM1, TPM2, TPM3, and TPM4) each of which generate a multitude of alternatively spliced mRNAs. TPM isoform diversity in bovine unlike in humans are not well characterized. The purpose of this investigation is to perform an extensive analysis of the expression of both transcripts and corresponding protein of sarcomeric TPMs in bovine strated muscles. We have cloned and sequenced the transcripts of the sarcomeric isoform of the TPM4 gene designated as TPM4α as well as a new splice variant TPM4ε from bovine striated muscles. Additionally, we have determined the expression of various sarcomeric TPM isoforms and TPM4ε in bovine heart and skeletal muscles. Relative expression as well as absolute copy number determination by qRT-PCR suggests that TPM1α expression is significantly higher in bovine cardiac muscle, whereas TPM2α is higher in skeletal muscle. The relative expression of TPM3α in bovine heart and skeletal muscle is very similar. The relative expression of TPM4α and TPM4ε is higher in bovine heart and skeletal muscle, respectively. We have evaluated the protein expression levels of various TPM isoforms by 2D western blot analyses in commercially available protein extracts of heart and skeletal muscles with the CH1 monoclonal antibody against TPM. Protein from each CH1-positive spot was extracted for LC-MS/MS analyses, which show that bovine heart extract contains 91.66% TPM1 and 8.33% TPM2, whereas skeletal muscle extract contains 57% TPM1 and 42.87% TPM2. We have failed to detect the presence of unique peptide(s) for TPM3α, TPM4α, and TPM4ε.

Inhibitors of the ubiquitin proteasome system block myofibril assembly in cardiomyocytes derived from chick embryos and human pluripotent stem cells.

Details of sarcomeric protein assembly during de novo myofibril formation closely resemble myofibrillogenesis in skeletal and cardiac myocytes in birds, rodents, and zebrafish. The arrangement of proteins during myofibrillogenesis follows a three-step process: beginning with premyofibrils, followed by nascent myofibrils, and concluding with mature myofibrils (reviewed in Sanger et al., 2017). Assembly and maintenance of myofibrils in living muscle cells. In: Handbook of experimental pharmacology, 2017 [pp. 39-75]. Our aim is to determine if the same pathway is followed in human cardiomyocytes derived from human inducible pluripotent stem cells. We found that the human cardiomyocytes developed patterns of protein organization identical to the three-step series seen in the model organisms cited above. Further experiments showed that myofibril assembly can be blocked at the nascent myofibril by five different inhibitors of the Ubiquitin Proteasome System (UPS) stage in both avian and human cardiomyocytes. With the exception of Carfilzomib, removal of the UPS inhibitors allows nascent myofibrils to proceed to mature myofibrils. Some proteasomal inhibitors, such as Bortezomib and Carfilzomib, used to treat multiple myeloma patients, have off-target effects of damage to hearts in three to 6 % of these patients. These cardiovascular adverse events may result from prevention of mature myofibril formation in the cardiomyocytes. In summary, our results support a common three-step model for the formation of myofibrils ranging from avian to human cardiomyocytes. The Ubiquitin Proteasome System is required for progression from nascent myofibrils to mature myofibrils. Our experiments suggest a possible explanation for the cardiac and skeletal muscle off-target effects reported in multiple myeloma patients treated with proteasome inhibitors.

Pfizer-biontech COVID-19 RNA vaccination induces phosphatidylserine autoantibodies, cryoglobulinemia, and digital necrosis in a patient with pre-existing autoimmunity.

We describe a 64-year-old Caucasian female with a history of Raynaud's disease, hand arthritis, photosensitivity, Sjogren's syndrome and leukocytoclastic vasculitis who presented with progressively worsening fingertip necrosis that began three days after receiving a first dose of Pfizer-BioNTech COVID-19 RNA vaccine. Our workup revealed cryoglobulinemia, hypocomplementemia, elevated antinuclear antibodies (ANA) and IgM antiphospholipid autoantibodies (aPL) directed against phosphatidylserine (aPL-PS), suggesting a diagnosis of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). The patient failed to develop anti-spike IgG antibodies up to two months following vaccination. Disease progression was halted by plasmapheresis, anticoagulation, and immune suppression. We conclude that the vaccine RNA moiety may induce SLE manifesting in APS, cryoglobulinemia, hypocomplementemia, and digital necrosis.

Sarcomeric TPM3 expression in human heart and skeletal muscle.

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.

Retroviral sero-reactivity in LGL leukaemia patients and family members.

T-cell large granular lymphocyte (T-LGL) leukaemia is characterized by a clonal proliferation of cytotoxic T cells and is frequently associated with rheumatoid arthritis. Sera from some LGL leukaemia patients react to a portion of the human T-cell leukaemia virus (HTLV-1/2) transmembrane envelope protein, BA21, although HTLV-1/2 infection is rare in LGL leukaemia patients. Here we show that family members, including spouses, of an LGL leukaemia patient had elevated LGL counts, BA21 reactivity and, additionally, recognition of HIV-1 gp41. Thus, both LGL leukaemia patients and clinically normal contacts sharing the same environment have evidence of exposure to a retrovirus.

Qualitative and quantitative evaluation of TPM transcripts and proteins in developing striated chicken muscles indicate TPM4α is the major sarcomeric cardiac tropomyosin from early embryonic life to adulthood.

The chicken has been used since the 1980s as an animal model for developmental studies regarding tropomyosin isoform diversity in striated muscles, however, the pattern of expression of transcripts as well as the corresponding TPM proteins of various tropomyosin isoforms in avian hearts are not well documented. In this study, using conventional and qRT-PCR, we report the expression of transcripts for various sarcomeric TPM isoforms in striated muscles through development. Transcripts of both TPM1α and TPM1κ, the two sarcomeric isoforms of the TPM1 gene, are expressed in embryonic chicken hearts but disappear in post hatch stages. TPM1α transcripts are expressed in embryonic and adult skeletal muscle. The sarcomeric isoform of the TPM2 gene is expressed mostly in embryonic skeletal muscles. As reported earlier, TPM3α is expressed in embryonic heart and skeletal muscle but significantly lower in adult striated muscle. TPM4α transcripts are expressed from embryonic to adult chicken hearts but not in skeletal muscle. Our 2D Western blot analyses using CH1 monoclonal antibody followed by mass spectra evaluations found TPM4α protein is the major sarcomeric tropomysin expressed in embryonic chicken hearts. However, in 7-day-old embryonic hearts, a minute quantity of TPM1α or TPM1κ is also expressed. This finding suggests that sarcomeric TPM1 protein may play some important role in cardiac contractility and/or cardiac morphogenesis during embryogenesis. Since only the transcripts of TPM4α are expressed in adult chicken hearts, it is logical to presume that TPM4α is the only sarcomeric TPM protein produced in adult cardiac tissues.

Sarcomeric TPM3α in developing chicken.

Cloning and sequencing of various tropomyosin isoforms expressed in chickens have been described since the early 1980s. However, to the best of our knowledge, this is the first report on the molecular characterization and the expression of the sarcomeric isoform of the TPM3 gene in cardiac and skeletal muscles from developing as well as adult chickens. Expression of TPM3α was performed by conventional RT-PCR as well as qRT-PCR using relative expression (by ΔCT as well as ΔΔCT methods) and by determining absolute copy number. The results employing all these methods show that the expression level of TPM3α is maximum in embryonic (10-day/15-day old) skeletal muscle and can barely be detected in both cardiac and skeletal muscles from the adult chicken. Our various RT-PCR analyses suggest that the expression of high molecular weight TPM3 isoforms are regulated at the transcription level from the proximal promoter at the 5'-end of the TPM3 gene.

Is MMTV associated with human breast cancer? Maybe, but probably not.

BACKGROUND: Conflicting results regarding the association of MMTV with human breast cancer have been reported. Published sequence data have indicated unique MMTV strains in some human samples. However, concerns regarding contamination as a cause of false positive results have persisted. METHODS: We performed PCR assays for MMTV on human breast cancer cell lines and fresh frozen and formalin fixed normal and malignant human breast epithelial samples. Assays were also performed on peripheral blood mononuclear cells from volunteer blood donors and subjects at risk for human retroviral infections. In addition, assays were performed on DNA samples from wild and laboratory mice. Sequencing of MMTV positive samples from both humans and mice were performed and phylogenetically compared. RESULTS: Using PCR under rigorous conditions to prevent and detect "carryover" contamination, we did detect MMTV DNA in human samples, including breast cancer. However, the results were not consistent and seemed to be an artifact. Further, experiments indicated that the probable source of false positives was murine DNA, containing endogenous MMTV, present in our building. However, comparison of published and, herein, newly described MMTV sequences with published data, indicates that there are some very unique human MMTV sequences in the literature. CONCLUSION: While we could not confirm the true presence of MMTV in our human breast cancer subjects, the data indicate that further, perhaps more traditional, retroviral studies are warranted to ascertain whether MMTV might rarely be the cause of human breast cancer.

Expression of various sarcomeric tropomyosin isoforms in equine striated muscles.

In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.

Identification, characterization, and expression of sarcomeric tropomyosin isoforms in zebrafish.

Tropomyosin is a component of thin filaments that constitute myofibrils, the contractile apparatus of striated muscles. In vertebrates, except for fish, four TPM genes TPM1, TPM2, TPM3, and TPM4 are known. In zebrafish, there are six TPM genes that include the paralogs of the TPM1 (TPM1-1 and TPM1-2), the paralogs of the TPM4 gene (TPM4-1 and TPM4-2), and the two single copy genes TPM2 and TPM3. In this study, we have identified, cloned, and sequenced the TPM1-1κ isoform of the TPM1-1 gene and also discovered a new isoform TPM1-2ν of the TPM1-2. Further, we have cloned and sequenced the sarcomeric isoform of the TPM4-2 gene designated as TPM4-2α. Using conventional RT-PCR, we have shown the expression of the sarcomeric isoforms of TPM1-1, TPM1-2, TPM2, TPM3, TPM4-1, and TPM4-2 in heart and skeletal muscles. By qRT-PCR using both relative expression as well as the absolute copy number, we have shown that TPM1-1α, TPM1-2α, and TPM1-2ν are expressed mostly in skeletal muscle; the level of expression of TPM1-1κ is significantly lower compared to TPM1-1α in skeletal muscle. In addition, both TPM4-1α and TPM4-2α are predominantly expressed in heart. 2D Western blot analyses using anti-TPM antibody followed by Mass Spectrometry of the proteins from the antibody-stained spots show that TPM1-1α and TPM3α are expressed in skeletal muscle whereas TPM4-1α and TPM3α are expressed in zebrafish heart. To the best of our knowledge, this is by far the most comprehensive analysis of tropomyosin expression in zebrafish, one of the most popular animal models for gene expression study.

Delayed Seroconversion to HTLV-II Is Associated with a Stop-Codon Mutation in the pol Gene.

A known HIV-1-positive intravenous drug user was found to be human T cell lymphoma/leukemia virus-II (HTLV-II) DNA positive by polymerase chain reaction but seronegative in a screening ELISA. He was consistently DNA positive but took 2 years to fully seroconvert. Sequencing of the HTLV-II strain in his cultured T lymphocytes indicated that it is a prototypical type A strain with no major differences in the long terminal repeat DNA sequence, nor major amino acid differences in the Gag, Env, Tax, and Rex proteins. However, a mutation in its pol gene created a stop codon at amino acid 543 of the Pol protein, a region that encodes for the RNase function. This mutation may account for the subject's slow seroconversion.

Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans.

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot "G" reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

Expression of tropomyosin 2 gene isoforms in human breast cancer cell lines.

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 TPM2ß was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2ß 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 TPM2ß 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 TPM2ß may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells.

Efficacy and Toxicity of Every 2 Weeks Docetaxel Regimen in Comparison With Weekly or Every 3 Weeks in Metastatic Prostate Cancer: A Retrospective Analysis.

OBJECTIVES: The aim of this study was to compare weekly (q1w), 2 weekly (q2w), and 3 weekly (q3w) regimens of docetaxel in metastatic castration-resistant prostate cancer (CRPC). MATERIALS AND METHODS: We retrospectively studied patients treated with q1w, q2w, or q3w docetaxel regimens at 30, 60, and 75 mg/m, respectively. The choice and duration of treatment was decided by their oncologist. Patients were assessed for response, progression-free survival (PFS), and overall survival (OS), and toxicity. RESULTS: Twelve, 14, and 15 patients were in the q1w, q2w, and q3w arms, respectively. Patients' age, metastases, and mean prostate-specific antigen at start and nadir were similar among groups. Mean total dose (MTD) was higher (not significantly) in the q2w group. Response rates, mean, and median PFS and OS ranked q2w>q3w>q1w (not significantly). However, hazard ratios for PFS for the q2w and q3w arms were statistically superior to the q1w arm when adjusted for age and total dose. The same was true for OS when q3w was compared with q1w. There were no significant differences between the q2w and q3w arms. Toxicities were not different between any of the arms, save for grade 1/2 neuropathy (lower in q1w compared with q2w). CONCLUSIONS: The MTD, response rates, PFS, and OS in the q1w and q3w arms were similar to published reports. Although we had a small number of patients, our findings suggest that both dose concentration and total docetaxel dose may be important in the treatment of CRPC and q2w dosing is an option in patients intolerant of a higher dose concentration.

Expression of Tropomyosin 1 Gene Isoforms in Human Breast Cancer Cell Lines.

Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1γ and TPM1δ were the dominant transcripts of TPM1, there was no clear evidence for TPM1δ protein expression. Four novel human TPM1 gene RNA isoforms were discovered (λ, µ, ν, and ξ), which were not identified in adult and fetal human cardiac tissues. TPM1λ was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ, λ, and ν protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γ RNA expression but significantly and inversely correlated with TPM1δ and TPM1λ expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study.

Increased seroreactivity to human T cell lymphoma/leukemia virus-related endogenous sequence-1 Gag peptides in patients with human T cell lymphoma/leukemia virus myelopathy.

Previously, we had shown that although only 8% of patients with large granular lymphocytic leukemia (LGLL) were infected with human T cell lymphoma/leukemia virus (HTLV)-2, almost half had antibodies to HTLV Gag and Env peptides. Herein, we investigated whether this could be due to cross-reactive antibodies to two homologous peptides in the Gag protein of the endogenous retrovirus HTLV-related endogenous sequence-1 (HRES-1). In addition, we had previously shown that patients with HTLV neurodegenerative diseases had increased seroreactivity to homologous HERV-K10 endogenous retrovirus peptides. Hence, in this study we also examined whether these patients had increased seroreactivity to the aforementioned HRES-1 Gag peptides. Sera from 100 volunteer blood donors (VBD), 53 patients with LGLL, 74 subjects with HTLV-1 or 2 infection (58 nonmyelopathy and 16 myelopathy), and 83 patients with multiple sclerosis (MS) were evaluated. The HTLV-positive myelopathy (HAM) patients had a statistically increased prevalence of antibodies to both HRES-1 Gag peptides (81%) vs. the VBD (0%), LGLL patients (13%), and MS patients (1%), and the HTLV-positive nonmyelopathy subjects (21%). The data suggest that cross-reactivity to HRES-1 peptides could be involved in the pathogenesis of HAM. The difference between the VBD and LGLL patients was also statistically significant, also suggesting a possible association in a minority of patients.