A platelet protein biochip rapidly detects an Alzheimer's disease-specific phenotype.

Alzheimer's disease (AD), a multifactorial neurodegenerative condition caused by genetic and environmental factors, is diagnosed using neuropsychological tests and brain imaging; molecular diagnostics are not routinely applied. Studies have identified AD-specific cerebrospinal fluid (CSF) biomarkers but sample collection requires invasive lumbar puncture. To identify AD-modulated proteins in easily accessible blood platelets, which share biochemical signatures with neurons, we compared platelet lysates from 62 AD, 24 amnestic mild cognitive impairment (aMCI), 13 vascular dementia (VaD), and 12 Parkinson's disease (PD) patients with those of 112 matched controls by fluorescence two-dimensional differential gel electrophoresis in independent discovery and verification sets. The optimal sum score of four mass spectrometry (MS)-identified proteins yielded a sensitivity of 94 % and a specificity of 89 % (AUC = 0.969, 95 % CI = 0.944-0.994) to differentiate AD patients from healthy controls. To bridge the gap between bench and bedside, we developed a high-throughput multiplex protein biochip with great potential for routine AD screening. For convenience and speed of application, this array combines loading control-assisted protein quantification of monoamine oxidase B and tropomyosin 1 with protein-based genotyping for single nucleotide polymorphisms (SNPs) in the apolipoprotein E and glutathione S-transferase omega 1 genes. Based on minimally invasive blood drawing, this innovative protein biochip enables identification of AD patients with an accuracy of 92 % in a single analytical step in less than 4 h.

Vardenafil improves penile erection in type 2 diabetes mellitus patients with erectile dysfunction: role of tropomyosin.

INTRODUCTION: Evidences have been suggested that phosphodiesterase type 5 (PDE5) inhibition promotes vasculoprotective benefits in patients with cardiovascular diseases. AIM: The aim of this study is to analyze the systemic effect of PDE5 inhibition in type 2 diabetes mellitus patients with erectile dysfunction (ED) determining changes in the expression levels of plasma proteins. METHODS: Seventeen patients with controlled type 2 diabetes mellitus and ED were included in the study. Patients received vardenafil hydrochloride 20 mg on demand during 12 weeks. At the beginning and 12 weeks after vardenafil administration, plasma samples were collected and analyzed using proteomics. MAIN OUTCOME MEASURES: International Index of Erectile Function-Erectile Function Domain (IIEF-EFD) and plasma protein expression before and after vardenafil administration. Nitrate/nitrite release, PDE5, and soluble guanylate cyclase (sGC) expression and cyclic guanosine monophosphate (cGMP) content in cultured bovine aortic endothelial cells (BAECs). RESULTS: The IIEF-EFD score was markedly improved after 12 weeks of vardenafil administration. Plasma levels of alpha 1-antitrypsin isotypes 4 and 6 and ß-tropomyosin were decreased, whereas apolipoprotein AI isoype 5 was increased 12 weeks after vardenafil administration. Only ß-tropomyosin plasma levels were inversely correlated with IIEF-EFD score. Tropomyosin has been added to cultured BAECs and after 24 hours reduced the protein expression level of sGC-ß1 subunit and decreased the cGMP content. Tropomyosin did not modify PDE5 expression and nitric oxide release in BAECs as compared with control BAECs. Vardenafil (10 µg/mL) did not modify sGC-ß1 subunit expression in tropomyosin + vardenafil-incubated BAECs; however, vardenafil significantly reversed the reduction of cGMP content induced by tropomyosin. CONCLUSION: Vardenafil administration improved erectile functionality in controlled type 2 diabetes mellitus patients with ED, which was associated with reduction of circulating plasma ß-tropomyosin levels. Tropomyosin affected by itself the cGMP generating system suggesting a possible new mechanism involved in ED. Vardenafil reversed the reduction effect of cGMP content elicited by tropomyosin in BAECs.

[Identification of 70 kDa shrimp protein as a possible new allergen for shrimp allergy].

BACKGROUND: A large-scale study of causative allergen components of shrimp allergies has never been conducted in Japan. SUBJECTS: A total of 31 patients with shrimp allergy who were referred to the Kakogawa Prefectural Medical Center from January 2004 to August 2011 were enrolled in the study. Shrimp allergy was diagnosed according to the clinical symptoms, and positive prick testing using black tiger shrimp. METHODS: Serum-specific IgE to two preparations of shrimp allergens (shrimp: shrimp extracts used before June 2012; and new shrimp: shrimp extracts used after July 2012 for ImmunoCAP®) and tropomyosin was determined with ImmunoCAP® (CAP-FEIA, Phadia) . Western blot analysis was performed using soluble and insoluble fractions from black tiger shrimp to define the causative shrimp allergens. RESULTS: In 31 cases of shrimp allergy, detection rate (more than class 1) of allergen-specific IgE to conventional shrimp was 58.1%, to new shrimp was 66.7%, and to tropomyosin was 29.0%. Positive rate (more than class 2) of allergen-specific IgE to conventional shrimp was 54.8%, to new shrimp was 55.0%, and to tropomyosin was 19.4%. In the 5 cases of FDEIA, detection rate of allergen-specific IgE to conventional shrimp was 20%, to new shrimp was 40%, and to tropomyosin was 0%. In the 19 cases of immediate-type allergy, detection rate of allergen-specific IgE to conventional shrimp was 68.4%, to new shrimp was 66.7%, and to tropomyosin was 36.8%. In the 7 cases of OAS, detection rate of allergen-specific IgE to shrimp was 57.1%, to new shrimp was 85.7%, and to tropomyosin was 28.5%. Western blot analysis of the 31 cases showed that several cases showed a band with a molecular weight of 35-38 kDa, which corresponds to tropomyosin. However, a 70-kDa band was detected in 30 of 31 cases. CONCLUSION: The 70-kDa protein may be a new major allergen component of shrimp allergy.

Identification and validation of novel serum markers for early diagnosis of endometriosis.

BACKGROUND: Non-invasive diagnosis of endometriosis is urgently required to prevent the long delay between the onset of symptoms and diagnosis. A biomarker that possesses both high sensitivity and specificity is greatly required. Here, we describe the use of a proteomic approach to identify potential novel endometrial antigens using sera from endometriosis patients and healthy controls, with evaluation of biomarkers for non-invasive diagnosis of endometriosis. METHODS: A cross-sectional study was conducted to identify specific endometrial antigens using 1D and 2D western blots in women with early endometriosis (n = 17), advanced endometriosis (n = 23) and without endometriosis (n = 30). Five immunoreactive spots were analyzed using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry with MASCOT analysis. ELISAs were established for specific epitopes and autoantibody titres were estimated in an independent cohort comprising women with early endometriosis (n = 18), advanced endometriosis (n = 32) and without endometriosis (n = 27) for validation. RESULTS: The 2D western blot analysis resulted in the identification of three endometrial antigens, tropomyosin 3 (TPM3), stomatin-like protein 2 (SLP2) and tropomodulin 3 (TMOD3). Serum levels of antibodies against the epitopes from the immunodominant region of proteins TPM3, SLP2 and TMOD3 were significantly elevated in endometriosis patients when compared with controls. Sensitivity and specificity of serum anti-TPM3a-autoAb (61%, 93%), anti-TPM3c-autoAb (44%, 93%), anti-TPM3d-autoAb (78%, 89%), anti-SLP2a-autoAb (50%, 96%), anti-SLP2c-autoAb (61%, 93%), anti-TMOD3b-autoAb (61%, 96%), serum anti-TMOD3c-autoAb (78%, 93%) and anti-TMOD3d-autoAb (78%, 96%) were better than those of serum CA125 levels (21%, 89%) in the detection of early stages of endometriosis. CONCLUSIONS: Serum anti-TPM3a-autoAb, anti-TPM3c-autoAb, anti-TPM3d-autoAb, anti-SLP2a-autoAb, anti-SLP2c-autoAb, anti-TMOD3b-autoAb, anti-TMOD3c-autoAb and anti-TMOD3d-autoAb could be new markers for the early diagnosis of endometriosis.

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi.

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.

Detection of novel biomarkers of liver cirrhosis by proteomic analysis.

UNLABELLED: Hepatic cirrhosis is a life-threatening disease arising from different chronic liver disorders. One major cause for hepatic cirrhosis is chronic hepatitis C. Chronic hepatitis C is characterized by a highly variable clinical course, with at least 20% developing liver cirrhosis within 40 years. Only liver biopsy allows a reliable evaluation of the course of hepatitis C by grading inflammation and staging fibrosis, and thus serum biomarkers for hepatic fibrosis with high sensitivity and specificity are needed. To identify new candidate biomarkers for hepatic fibrosis, we performed a proteomic approach of microdissected cirrhotic septa and liver parenchyma cells. In cirrhotic septa, we detected an increasing expression of cell structure associated proteins, including actin, prolyl 4-hydroxylase, tropomyosin, calponin, transgelin, and human microfibril-associated protein 4 (MFAP-4). Tropomyosin, calponin, and transgelin reflect a contribution of activated stellate cells/myofibroblasts to chronic liver injury. The expression of tropomyosin, transgelin, and MFAP-4, an extracellular matrix associated protein, were further evaluated by immunohistochemistry. Tropomyosin and MFAP-4 demonstrated high serum levels in patients with hepatic cirrhosis of different causes. CONCLUSION: A quantitative analysis of MFAP-4 serum levels in a large number of patients showed MFAP-4 as novel candidate biomarker with high diagnostic accuracy for prediction of nondiseased liver versus cirrhosis [area under receiver operating characteristic curve (AUC) = 0.97, P < 0.0001] as well as stage 0 versus stage 4 fibrosis (AUC = 0.84, P < 0.0001), and stages 0 to 3 versus stage 4 fibrosis (AUC = 0.76, P < 0.0001).

Gold-microrods/Pd-nanoparticles/polyaniline-nanocomposite-interface as a peroxidase-mimic for sensitive detection of tropomyosin.

In this study, Gold-microrods (AuMRs), Pd-nanoparticles (PdNPs), and Polyaniline (PANI) nanocomposite-interface was fabricated on the screen-printed carbon-microelectrode (SPE). Each layer of the interface was characterised using field emission-scanning electron microscopy (FE-SEM) and cyclic voltammetry (CV). The fabricated SPE/AuMRs/PdNPs/PANI interface demonstrated the highest electronic current and showed excellent peroxidase-mimic towards H2O2 using chronoamperometry (CA). Furthermore, the SPE/AuMRs/PdNPs/PANI interface was utilised for the construction of a highly sensitive label-free electrochemical biosensor for the detection of Tpm in seafood samples. Label-free electrochemical detection of the Tpm was performed using both CA and differential pulse voltammetry (DPV) techniques. Preliminary data showed that both methods could detect Tpm as low as 0.01 pg/mL. Moreover, the developed biosensor for the detection of Tpm demonstrated excellent selectivity, high reproducibility and longer stability with an evident potential to detect Tpm in real seafood samples.

Proteomic analysis of mononuclear cells of patients with minimal-change nephrotic syndrome of childhood.

UNLABELLED: Background/Aims. Recently, peripheral blood mononuclear cell transcriptome analysis has identified genes that are upregulated in relapsing minimal-change nephrotic syndrome (MCNS). In order to investigate protein expression in peripheral blood mononuclear cells (PBMC) from relapsing MCNS patients, we performed proteomic comparisons of PBMC from patients with MCNS in relapse and controls. METHODS: PBMC from a total of 20 patients were analysed. PBMC were taken from five patients with relapsing MCNS, four in remission, five patients with other glomerular diseases and six controls. Two dimensional electrophoresis was performed and proteome patterns were compared. RESULTS: Automatic heuristic clustering analysis allowed us to pool correctly the gels from the MCNS patients in the relapse and in the control groups. Using hierarchical population matching, nine spots were found to be increased in PBMC from MCNS patients in relapse. Four spots were identified by mass spectrometry. Three of the four proteins identified (L-plastin, alpha-tropomyosin and annexin III) were cytoskeletal-associated proteins. Using western blot and immunochemistry, L-plastin and alpha-tropomyosin 3 concentrations were found to be enhanced in PBMC from MCNS patients in relapse. Conclusions. These data indicate that a specific proteomic profile characterizes PBMC from MCNS patients in relapse. Proteins involved in PBMC cytoskeletal rearrangement are increased in relapsing MCNS. We hypothesize that T-cell cytoskeletal rearrangement may play a role in the pathogenesis of MCNS by altering the expression of cell surface receptors and by modifying the interaction of these cells with glomerular cells.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

BACKGROUND: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. METHODS: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. RESULTS: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. CONCLUSIONS: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

Identification by serological proteome analysis of paramyosin as prominent allergen in dust mite allergy.

Component-resolved diagnosis (CRD) of IgE-mediated hypersensitivities is challenged by the possibility that single patients are sensitized to components not commercially available to the clinical lab. Here, we studied a patient with positive extract-based diagnosis of house dust mite (HDM) allergy based on routine in vivo (prick test) and in vitro (serum specific IgE) tests, whose serum scored negative for IgE to the three recombinant allergens routinely used in CRD (group 1 allergens, group 2 allergens and tropomyosin). By means of serological proteome analysis via two-dimensional gel electrophoresis combined with immunoblotting and mass spectrometry, paramyosin (group 11 allergen: Der f 11 and Der p 11) was identified as the allergen component recognized by serum IgE from this patient in a raw allergen extract. Nine patients (64%) had IgE to Der p 11 in a group of 14 HDM allergic patients. BIOLOGICAL SIGNIFICANCE: Our results add up to previous reports indicating that paramyosin is a clinically relevant HDM allergen and highlight that it can represent, in some patients, the first sensitizing component of this allergen source. This suggests that, at the moment, the use of allergen extract for the purpose of measuring IgE reactivity cannot be replaced by component resolved diagnosis and that group 11 allergens should be included among allergen components routinely tested in the clinical laboratory.

Stabilization of F-actin by tropomyosin isoforms regulates the morphology and mechanical behavior of red blood cells.

The short F-actins in the red blood cell (RBC) membrane skeleton are coated along their lengths by an equimolar combination of two tropomyosin isoforms, Tpm1.9 and Tpm3.1. We hypothesized that tropomyosin's ability to stabilize F-actin regulates RBC morphology and mechanical properties. To test this, we examined mice with a targeted deletion in alternatively spliced exon 9d of Tpm3 (Tpm3/9d-/- ), which leads to absence of Tpm3.1 in RBCs along with a compensatory increase in Tpm1.9 of sufficient magnitude to maintain normal total tropomyosin content. The isoform switch from Tpm1.9/Tpm3.1 to exclusively Tpm1.9 does not affect membrane skeleton composition but causes RBC F-actins to become hyperstable, based on decreased vulnerability to latrunculin-A-induced depolymerization. Unexpectedly, this isoform switch also leads to decreased association of Band 3 and glycophorin A with the membrane skeleton, suggesting that tropomyosin isoforms regulate the strength of F-actin-to-membrane linkages. Tpm3/9d-/- mice display a mild compensated anemia, in which RBCs have spherocytic morphology with increased osmotic fragility, reduced membrane deformability, and increased membrane stability. We conclude that RBC tropomyosin isoforms directly influence RBC physiology by regulating 1) the stability of the short F-actins in the membrane skeleton and 2) the strength of linkages between the membrane skeleton and transmembrane glycoproteins.

Panel of Autoimmune Markers for Noninvasive Diagnosis of Minimal-Mild Endometriosis.

Endometriosis, characterized by the presence of endometrial-like tissue at extrauterine sites, is a common, chronic, estrogen-dependent, inflammatory condition associated with pelvic pain, subfertility, dysmenorrhea, and dyspareunia, affecting about 10% of reproductive-age women in any population. The diagnosis of endometriosis is usually delayed on an average by 8 to 11 years leading to significant consequences in terms of disease progression. The current study was aimed to validate enzyme-linked immunosorbent assay based on the epitopes of stomatin-like protein 2, tropomodulin 3 (TMOD3), and tropomyosin 3 (TPM3) for diagnosis of minimal-mild endometriosis (revised American Fertility Society Classification (rAFS) stage I-II) and to compare the performance with the reported markers: cancer antigen (CA) 125, CA19-9, α-enolase, Serine/threonine-protein kinase (PDIK1L), and syntaxin 5. This was a cross-sectional, multicenter study conducted during the year 2012 to 2015. Women with minimal-mild endometriosis (rAFS stage I-II [n = 133]) and healthy controls (n = 104) were screened for 11 novel autoimmune markers and reported markers α-enolase, PDIK1L, syntaxin 5, CA-125, and CA19-9. The sensitivity and diagnostic accuracy of serum antibodies against all the 11 epitopes were higher than that of CA-125, CA19-9, α-enolase, PDIK1L, and syntaxin 5 for diagnosis of rAFS stage I to II endometriosis. The sensitivity of 6 biomarkers (anti-TMOD3b-autoAb, anti-TMOD3c-autoAb, anti-TMOD3d-autoAb, anti-TPM3a-autoAb, anti-TPM3c-autoAb, and anti-TPM3d-autoAb) was higher at the specificity of ≥80% for diagnosis of rAFS stage I to II endometriosis as well as ultrasound-negative endometriosis. Further, logistic regression models of this panel of biomarkers showed increase in sensitivity, specificity, and diagnostic accuracy than individual biomarkers. The panel of 6 autoimmune biomarkers could be useful in setting up of noninvasive diagnostic test for detection of minimal-mild endometriosis.

Paramyosin of canine Onchocerca lupi: usefulness for the diagnosis of a neglected zoonotic disease.

BACKGROUND: Of increasing importance to the medical and veterinary communities is the zoonotic filarioid nematode Onchocerca lupi. Onchocercosis, thus far found in wolves, dogs, cats and humans, is diagnosed via skin snips to detect microfilariae and surgical removal of adults from the eye of the host. These methods are time-consuming, laborious and invasive, highlighting the need for new tools for the diagnosis of O. lupi in susceptible hosts. Symptoms related to the presence of the adults in the eye can range from none apparent to severe, including blindness. No reliable chemotherapeutic protocols are available, as yet, to eliminate the infection. Paramyosin, an invertebrate-specific protein, has been well-studied as an allergen, diagnostic marker and vaccine candidate. The aim of this study, therefore, was to isolate and characterise paramyosin from O. lupi to assess its suitability for the development of a serological diagnostic assay. METHODS: The adult and microfilarial stages of O. lupi were isolated from the eyes and skin of a 3-year-old male dog. Total RNA was extracted and reverse transcribed into single stranded cDNA. Reverse-transcription PCR was used to isolate a full-length paramyosin cDNA from adult worms and to investigate the temporal expression patterns of this gene. All amplicons were sequenced using dideoxy chain termination sequencing. Bioinformatics was used to predict the amino acid sequence of the gene, to compare the DNA and protein sequences with those available in public databases and to investigate the phylogenetic relationship of all molecules. Antibody binding sites were predicted using bioinformatics and mapped along with published antigenic epitopes against the O. lupi paramyosin protein. The native protein, and three smaller recombinantly expressed peptides, were subjected to western blot using serum from dogs both positive and negative for O. lupi. RESULTS: Paramyosin of O. lupi was herein molecularly characterized, encoded by a transcript of 2,643 bp and producing a protein of 881 amino acids (101.24 kDa). The paramyosin transcript was detected, by reverse transcription PCR, in adults and microfilariae, but not in eggs. Phylogenetic analysis indicates that this molecule clusters with paramyosins from other filarioids to the exclusion of those from other taxa. A total of 621 unique antibody binding epitopes were predicted for this protein and another 28 were conserved in other organisms. This information was used to design three peptides, for recombinant expression, to identify the antibody binding epitope(s) and reduce potential cross-reactivity with serum from dogs infected with other filarioid nematodes. Native paramyosin, purified from microfilariae and adults, was detected by antibodies present in serum from dogs with known O. lupi infections. CONCLUSIONS: Data provided herein may assist in the development of a serological diagnostic test, based on antibodies to O. lupi paramyosin, for the diagnosis of this infection, in order to gain more information on the real distribution of this little known filarioid of zoonotic concern.

Tropomyosin from human erythrocyte membrane polymerizes poorly but binds F-actin effectively in the presence and absence of spectrin.

Actin in the human erythrocyte forms short protofilaments which are only long enough to accommodate tropomyosin monomers (Shen, B.W., Josephs, R. and Steck, T.L. (1986) J. Cell Biol. 102, 997-1006). This interaction between actin and tropomyosin monomers is predicted to be weak, since tropomyosin polymerization parallels its affinity for F-actin. We examine the binding of human erythrocyte tropomyosin to actin in the presence and absence of spectrin and its ability to polymerize. The binding of human erythrocyte tropomyosin to F-actin is not affected appreciably by the present of spectrin. Saturating F-actin with erythrocyte tropomyosin, however, weakens the binding of spectrin dimers to actin. Although tropomyosin from human erythrocyte and rabbit cardiac muscle have similar affinity for F-actin, the polymerizability of erythrocyte tropomyosin as determined by viscosity measurements is much reduced relative to muscle tropomyosin. This unusual property of erythrocyte tropomyosin is likely due to differences in its primary structure from other known tropomyosin at the amino and carboxyl terminal regions which are responsible for its head-to-tail polymerization and cooperative binding to F-actin. Analysis of the distribution of tyrosine by 2-dimensional tryptic mapping of 125I-labelled erythrocyte tropomyosin shows that tyrosine at positions 162, 214, 221, 261 and 267 in rabbit cardiac tropomyosin are conserved in human erythrocyte tropomyosin but Tyr-60 is absent. This observation suggests that erythrocyte tropomyosin has a carboxyl terminal region similar to its muscle counterparts but its amino terminal region resembles that of platelet tropomyosin which also lacks Tyr-60.

Troponins: current status in coronary artery disease.

The acute coronary syndromes (ACS) represent a pathological, diagnostic and risk continuum from unstable angina (UA) through myocardial infarction (MI) with or without ST segment elevation. The past 12 years have seen extensive investigations into the use of various cardiac markers to establish the diagnosis and prognosis in ACS and to evaluate perfusion after thrombolysis. The Troponins comprise a group of three proteins (C, I and T) which interact with tropomyosin to form a troponin-tropomyosin complex. Troponin-T is a structural component of the troponin complex and is known to exist in three isoforms. Troponins have both diagnostic, post-event risk stratification and prognostic significance. Apart from myocardial infarction however they are raised in several conditions like Myocarditis, dilated Cardiomyopathy, Severe congestive cardiac failure, severe pulmonary embolism with right ventricular strain and Preterm infants with respiratory distress. False positives have been reported with Angioplasty, cardiac surgery, RF ablation, Allograft rejection following cardiac transplant and seropositive rheumatoid arthritis. False negatives have been reported with early sampling, early reading, use of wrong anticoagulant, clotted blood, careless storage of kits. Significantly, lower troponin values have been reported in heparinised plasma than in serum.

The cytoskeleton of blood platelets viewed by immunofluorescence microscopy.

Immunofluorescence microscopy has been used to characterize the morphological transitions that occur as platelets spread on a surface. Antibodies to the microfilament-specific proteins, actin, myosin, tropomyosin, alpha-actinin and filamin as well as antibodies to tubulin were used. Antibody to tubulin reveals the marginal band of microtubules as a bright fluorescent ring, the diameter of which decreases at a time coincident with pseudopod formation. The latter process is dictated by the assembly of microfilament bundles. Although the change in morphology of the platelet was not studied in detail, our data support the idea that microfilament reorganization influences the display of the marginal band of microtubules. A further conclusion is that the platelet in spite of its small diameter is a system suitable for immunofluorescence microscopy, a method which allows the rapid and simultaneous screening of many cells.

Amino acid sequence of equine platelet tropomyosin. Correlation with interaction properties.

Equine platelet beta tropomyosin (247 residues), like rabbit skeletal muscle alpha tropomyosin (284 residues) has a repeating pattern of amino acid residues characteristic of a coiled-coil structure. When compared with the muscle protein, it is extended by 5 residues at the NH2-terminus and possesses two 21 residue deletions (positions 23-43 and 60-80 of the muscle sequence). The two proteins are highly conserved from residues 81-260, but are significantly different at their COOH-termini (residues 261-284). These differences in platelet tropomyosin can be correlated with its diminished head-to-tail polymerization, a weaker interaction with F-actin and a reduced affinity for muscle troponin and the T1 fragment of troponin-T.

Abnormal thiol reactivity of tropomyosin in essential hypertension and its association with abnormal sodium-lithium countertransport kinetics.

OBJECTIVES: To identify a thiol protein that is abnormal in a subgroup of essential hypertensive (EHT) patients who have a strong family history of hypertension and cardiovascular disease and have a low Km of erythrocyte Na/Li countertransport (CT). METHODS: To detect biotin maleimide labelling of a key thiol protein to investigate its reaction with N-ethylmaleimide (NEM) in normal and EHT erythrocytes. RESULTS: The thiol protein of 33 kDa apparent molecular weight (p33) identified by the loss of labelling with biotin maleimide was identified as tropomyosin due to its retarded running in 6 mol/l urea gels and immunoblotting. The NEM reaction with p33 detected by loss of subsequent biotin maleimide labelling is biphasic in normal control erythrocytes with the rate in the first 30 s double that after 30 s. In EHT erythrocytes NEM reaction (1) after 30 s is faster than normal and (2) in the first 30 s causes a paradoxical increase in apparent biotin maleimide labelling. In normal control erythrocytes, the loss of biotin maleimide labelling with NEM reaction or the faster phenylmaleimide reaction follows the same time course as the decrease in Km of Na/Li CT. CONCLUSIONS: NEM reaction with p33 requires two thiols. Only the cytoskeletal form of tropomyosin from the TM3 gene has more than one thiol group and agrees with SDS-PAGE mobility. Tropomyosin is a strong candidate to explain the familial abnormality in EHT with abnormal Na/ Li CT and it could explain many of the characteristics of this disease.

Naturally occurring IgG autoantibodies to platelet cytoskeleton tropomyosin.

We have observed that naturally occurring serum antibodies generated a 30 Kd band in a platelet immunoblot assay. The target protein had the same molecular weight (30 Kd) under nonreduced and reduced electrophoretic conditions, and could be immunoblotted from either autologous or homologous platelet lysates. Also, the 30 Kd reactive autoantibodies could be totally adsorbed by platelet cytoskeletons. From these data one likely candidate for the autoantibody target was the intracellular platelet protein tropomyosin. Indeed, a commercially available monoclonal anti-tropomyosin antibody reacted with proteins comigrating with this 30 Kd band; affinity purified human platelet tropomyosin was bound by the antibodies that recognized the 30 Kd protein. This body of evidence conclusively demonstrated that naturally occurring serum autoantibodies reacted with the platelet cyto-skeleton protein-tropomyosin. These tropomyosin specific antibodies were found in roughly the same percentage of sera from patients with chronic idiopathic thrombocytopenic purpura (ITP) as from normal individuals.

Clinical significance of elevated troponin I levels in patients with nontraumatic subarachnoid hemorrhage.

OBJECT: Aneurysmal subarachnoid hemorrhage (SAH) is associated with electrocardiographic abnormalities, regional or focal wall-motion abnormalities on echocardiograms, and/or increased creatine kinase MB isoenzyme (CK-MB) or cardiac troponin I (cTnI). The goal of this prospective study was to compare the sensitivity and specificity of cTnI with those of CK-MB in the prediction of left ventricular dysfunction on echocardiograms in patients with nontraumatic SAH. In addition, those patients with abnormal findings on their echocardiograms and elevated cTnI levels were further evaluated for the presence of coronary artery disease (CAD) by a cardiologist and to determine whether any left ventricular dysfunction that had been detected was reversible. METHODS: The authors obtained electrocardiograms and echocardiograms, and measured serial levels of cardiac enzymes (CK-MB and cTnI) in 43 patients with nontraumatic SAH. Patients with known CAD were excluded. Those patients found to have elevated enzyme levels and abnormal findings on their echocardiograms underwent additional evaluation for CAD. The sensitivity and specificity of both cTnI and CK-MB for detecting left ventricular function were determined. Twenty-eight percent of patients with SAH in the study had elevated cTnI levels within the first 24 hours after hemorrhage. Seven of the 12 patients had evidence of left ventricular dysfunction on echocardiograms. In all these patients a return to baseline function was found during follow-up examinations. The authors found that cTnI is much more sensitive than CK-MB (100% compared with 29%) in the detection of left ventricular dysfunction in patients with SAH. CONCLUSIONS: An elevated level of cTnI is a good indicator of left ventricular dysfunction in patients with SAH. In this study cardiac dysfunction was reversible and should not necessarily preclude these patients from undergoing operative interventions or becoming heart donors. Clinical management may require more aggressive hemodynamic monitoring until cardiac function returns to normal.