Environmental enrichment changes the effects of prenatal and postnatal undernutrition on memory, anxiety traits, Bdnf and TrkB expression in the hippocampus of male adult rats.

Environmental factors such as undernutrition and environmental enrichment can promote changes in the molecular and behavioural mechanisms related to cognition. Herein, we investigated the effect of enriched environment stimulation in rats that were malnourished in the pre- and postnatal periods on changes in the gene expression of brain-derived neurotrophic factor and its receptor in the hippocampus, as well as on anxiety traits and memory. Early undernutrition promoted weight reduction, increased the risk analysis, reduced permanence in the open arm of the elevated plus-maze and induced a reduction in the gene expression of brain-derived neurotrophic factor and tropomyosin receptor kinase B. However, exposure to an enriched environment from 30 to 90 days' old maintained the malnourished phenotype, leading to weight reduction in the control group. In addition, the enriched environment did not alter the risk assessment in the undernourished group, but it did increase the frequency of labyrinth entries. Sixty-day exposure to the enriched environment resulted in a reversal in the gene expression of brain-derived neurotrophic factor and tropomyosin receptor kinase B in the hippocampus of malnourished rats and favoured of long-term memory in the object recognition test in the open-field. These results suggest that an enriched environment may have a protective effect in adult life by inducing changes in long-term memory and anxiety traits in animals that were undernourished in early life. Furthermore, reversing these effects of undernutrition involves mechanisms linked to the molecular signalling of brain-derived neurotrophic factor and tropomyosin receptor kinase B in the hippocampus.

Beta-caryophyllene mitigates the cognitive impairment caused by repeated exposure to aspartame in rats: Putative role of BDNF-TrKB signaling pathway and acetylcholinesterase activity.

Aspartame (ASP) is a common sweetener, but studies show it can harm the nervous system, causing learning and memory deficits. ß-caryophyllene (BCP), a natural compound found in foods, including bread, coffee, alcoholic beverages, and spices, has already described as a neuroprotector agent. Remarkably, ASP and BCP are commonly consumed, including in the same meal. Therefore, considering that (a) the BCP displays plenty of beneficial effects; (b) the ASP toxicity; and (c) that they can be consumed in the same meal, this study sought to investigate if the BCP would mitigate the memory impairment induced by ASP in rats and investigate the involvement of the brain-derived neurotrophic factor (BDNF)/ tropomyosin receptor kinase B (TrKB) signaling pathway and acetylcholinesterase (AChE) activity. Young male Wistar rats received ASP (75 mg/kg; i.g.) and/or BCP (100 mg/kg; i.p.) once daily, for 14 days. At the end of the treatment, the animals were evaluated in the open field and object recognition tests. The cerebral cortex and hippocampus samples were collected for biochemical and molecular analyses. Results showed that the BCP effectively protected against the cognitive damage caused by ASP in short and long-term memories. In addition, BCP mitigated the increase in AChE activity caused by ASP. Molecular insights revealed augmented BDNF and TrKB levels in the hippocampus of rats treated with BCP, indicating greater activation of this pathway. In conclusion, BCP protected against ASP-induced memory impairment. AChE activity and the BDNF/TrkB signaling pathway seem to be potential targets of BCP modulatory role in this study.

Upregulation of tropomyosin alpha-4 chain in patients' saliva with oral squamous cell carcinoma as demonstrated by Phage display.

Patients with oral squamous cell carcinoma (OSCC) present significant alterations in their saliva proteome. We have used the shotgun Phage Display (PD) technology to identify candidate proteins that were upregulated in saliva of OSCC by selecting ligands to salivary proteins from a single-chain variable fragment (scFv) PD combinatorial library. After two selection cycles, the highly reactive clone scFv-D09 was able to distinguish saliva of OSCC patients from healthy subjects by enzyme-linked immunosorbent assay (ELISA) with sensitivity and specificity of 96.67%. Additionally, the scFv-D09 clone presented a positive immunostaining for invasive malignant epithelial cells in the connective tissue, keratin pearls in the OSCC, and ducts of salivary glands. We have further identified the target protein as the tropomyosin alpha-4 chain (TPM4) by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, and its binding to the scFV-D09 was demonstrated by bioinformatics. Briefly, we have identified TPM4 as upregulated salivary protein in patients with OSCC, which plays a central role in stabilizing cytoskeleton actin filaments, probably linked with tumor tissue remodeling. Long-term longitudinal studies are needed to validate TPM4 as a potential marker of a malignant process.

Myofibrilar functional dysregulation in fish: A new biomarker of damage to pesticides.

Endosulfan (ES) modifies the ultrastructure of skeletal muscle fibers and causes changes to the swimming behavior of fish. The objectives of the present work were to evaluate, in fishes of Australoheros facetus, 1) the integrity of myofibrils (Mf) by the analysis of SDS-PAGE profiles, and 2) the functionality of Mf through the microscopically monitoring of the contraction and changes in Mg2+ Ca2+- ATPase and Mg2+(EGTA) -ATPase activities. As expected, after the addition of the contraction buffer, control fish Mf contracted. On the contrary, Mf from fish exposed at 0.5 µg/L ES showed a partial contraction and none of the fish exposed at 10 µg/L ES contracted. As judged by its high Mg2+ Ca2+ ATPase activity and low Mg2+ (EGTA) ATPase activity, control Mf showed good functionality. In Mf from fish exposed to 0.5 and 10 µg/L ES the activities of these enzymes were similar, suggesting denaturation or degradation of some component of tropomyosin-troponin complex. SDS-PAGE patterns of Mf from fish exposed to ES showed degradation of the myosin heavy chain and of tropomyosin. Similar values of lipid peroxidation (TBARS) were found in both control and exposed Mf, suggesting that lipid oxidation was not be related to the above-mentioned changes. The observed effects expand the knowledge of ES action in muscles and could be used as biomarkers of damage in fishes exposed to organochlorine compounds like the insecticide endosulfan.

Evaluation of the immunoreactivity of nerve growth factor and tropomyosin receptor kinase A in the esophagus of noninfected and infected individuals with Trypanosoma cruzi.

Megaesophagus is one of the major manifestations of the chronic phase of Chagas disease. Its primary symptom is generally dysphagia due to disturbance in the lower esophageal sphincter. Microscopically, the affected organ presents denervation, which has been considered as consequence of an inflammatory process that begins at the acute phase and persists in the chronic phase. Inflammatory infiltrates are composed of lymphocytes, macrophages, natural killer cells, mast cells, and eosinophils. In this study, we evaluated the immunoreactivity of nerve growth factor (NGF), and of its receptor tropomyosin receptor kinase A (TrkA), molecules that are well known for having a relevant role in neuroimmune communication in the gastrointestinal tract. Esophageal samples obtained via autopsy or surgery procedures from six noninfected individuals, six infected individuals without megaesophagus, and six infected individuals with megaesophagus were analyzed. Infected individuals without megaesophagus presented increased numbers of NGF immunoreactive (IR) mast cells and increased areas of TrkA-IR epithelial cells and inner muscle cells. Infected individuals with megaesophagus showed increased numbers of NGF-IR eosinophils and mast cells, TrkA-IR eosinophils and mast cells, increased area of NGF-IR epithelial cells, and increased areas of TrkA-IR epithelials cells and inner muscle cells. The data presented here point to the participation of NGF and its TrkA receptor in the pathology of chagasic megaesophagus.

Physicochemical characterization of actomyosin-paramyosin from giant squid mantle (Dosidicus gigas).

BACKGROUND: The giant squid (Dosidicus gigas) has been proposed as raw material to obtain myofibrillar protein concentrates. However, it has been observed that colloidal systems formed from squid proteins have limited stability. Therefore, the isolation and characterization of the actomyosin-paramyosin isolated (API) complex were performed, because they are the main proteins to which functionality has been attributed. RESULTS: Densitogram analysis revealed 45% of actin, 38% of myosin and 17% of paramyosin. The amino acid profile indicates a higher proportion of acidic amino acids, which gives a higher negative charge; this was supported by the zeta potential. Total sulfhydryl (TSH) content was lower compared with proteins of other aquatic species. CONCLUSION: The higher percentage of actin in relation to myosin, the presence of paramyosin, as well as the low content of sulfhydryl groups, could comprise the main causes of the low technological functional property of proteins from D. gigas mantle. © 2017 Society of Chemical Industry.

Identification and Characterization of IgE-Binding Tropomyosins in Aedes aegypti.

BACKGROUND: The mosquito Aedes aegypti is a potential source of important clinically relevant allergens. However, the allergenicity and cross-reactivity of most of these has not been fully described. METHODS: Natural wild-type mosquito tropomyosin was purified by size exclusion and anionic-exchange chromatography from an A. aegypti extract. Further characterization was accomplished by MALDI-TOF/TOF. Two recombinant variants of tropomyosin were obtained by expression in Escherichia coli. Specific IgE measurement by ELISA and skin tests for mosquito extract were performed in 12 patients with asthma or allergy rhinitis residing on the Caribbean island of Martinique. Cross-reactivity between natural A. aegypti tropomyosin and recombinant tropomyosins from A. aegypti, house dust mite, shrimp and Ascaris lumbricoides was analyzed by ELISA competition. RESULTS: Four variants of natural tropomyosin were purified. A band of 32 kDa in SDS-PAGE representing 2 tropomyosin variants (Aed a 10.0101 and Aed a 10.0201) reacted with specific IgE of 4 of the 12 (33%) allergic patients and with rabbit polyclonal anti-shrimp tropomyosin. A high degree of cross-reactivity (60-70%) was detected between natural mosquito tropomyosin and Blo t 10, Der p 10 and Lit v 1, and a lower degree with Asc l 3 from A. lumbricoides (<30%). rAed a 10.0101 inhibited IgE binding to natural A. aegypti tropomyosin; however, rAed a 10.0201 showed a low inhibitory capacity. CONCLUSION: Tropomyosin is a new IgE-binding protein from A. aegypti. Two of the 4 variants identified showed different degree of cross-reactivity with tropomyosins from other arthropods. The potential allergenic role of each variant should be further investigated.

In vitro effects of 1α,25(OH)2D3-glycosides from Solbone A (Solanum glaucophyllum leaves extract; Herbonis AG) compared to synthetic 1α,25(OH)2D3 on myogenesis.

The presence of glycoside derivatives of 1α,25(OH)2D3 endows plants to gradual release of the free bioactive form of 1α,25(OH)2D3 from its glycoconjugates by endogenous animal tissue glycosidases. This results in increased half-life of the hormone in blood when purified plant fractions are administered for therapeutic purposes. In this work, we evaluated the role 1α,25(OH)2D3-glycosides enriched natural product (Solbone A) from Solanum glaucophyllum leaf extract compared with synthetic 1α,25(OH)2D3 on myogenic differentiation in C2C12 myoblasts. For these, differentiation markers and myogenic parameters were studied in C2C12 myoblasts. Results showed that Solbone A, likewise the synthetic hormone, increased creatine kinase activity at day 2 after differentiation induction (60%, p<0.05). Solbone A and synthetic 1α,25(OH)2D3 increased vitamin D3 receptor protein expression at 10nM (50% and 30%, respectively) and the transcription factor myogenin (80%, p<0.05). However, tropomyosin expression was not affected by both compounds. In addition, myosin heavy chain (MHC) protein expression was increased 30% at day 2 of differentiation. Solbone A or synthetic 1α,25(OH)2D3 had no effects on myogenin nor MHC cell localization. Cellular mass increased with myogenesis progression, being Solbone A more effective than synthetic 1α,25(OH)2D3. Finally, Solbone A, as well as synthetic 1α,25(OH)2D3, augmented the index fusion of cultured muscle fibers. In conclusion, these results demonstrated that Solbone A exhibit at least equal or greater effects on early myoblast differentiation as synthetic hormone, suggesting that plant glycosides could be an effective, accessible and cheaper substitute for synthetic 1α,25(OH)2D3 to promote muscle growth.

Absence of mutation at the 5'-upstream promoter region of the TPM4 gene from cardiac mutant axolotl (Ambystoma mexicanum).

Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

Membrane cholesterol depletion by methyl-beta-cyclodextrin enhances the expression of cardiac differentiation markers.

Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-beta-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/beta-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. beta-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac beta-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.

Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa.

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

Ultrastructural features of the columellar muscle and contractile protein analyses in different muscle groups of Megalobulimus abbreviatus (Gastropoda, Pulmonata).

In Megalobulimus abbreviatus, the ultrastructural features and the contractile proteins of columellar, pharyngeal and foot retractor muscles were studied. These muscles are formed from muscular fascicles distributed in different planes that are separated by connective tissue rich in collagen fibrils. These cells contain thick and thin filaments, the latter being attached to dense bodies, lysosomes, sarcoplasmic reticulum, caveolae, mitochondria and glycogen granules. Three types of muscle cells were distinguished: T1 cells displayed the largest amount of glycogen and an intermediate number of mitochondria, suggesting the highest anaerobic metabolism; T2 cells had the largest number of mitochondria and less glycogen, which suggests an aerobic metabolism; T3 cells showed intermediate glycogen volumes, suggesting an intermediate anaerobic metabolism. The myofilaments in the pedal muscle contained paramyosin measuring between 40 and 80nm in diameter. Western Blot muscle analysis showed a 46-kDa band that corresponds to actin and a 220-kDa band that corresponds to myosin filaments. The thick filament used in the electrophoresis showed a protein band of 100kDa in the muscles, which may correspond to paramyosin.

Mg2+ ions bind at the C-terminal region of skeletal muscle alpha-tropomyosin.

Tropomyosin (Tm) is a dimeric coiled-coil protein that polymerizes through head-to-tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg(2+)-induced increase in stability of the C-terminal half of Tm is sensitive to mutations near the C-terminus. In the present report, we study the interaction between Mg(2+) and full-length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm(259-284(W269))) is flexible and to large extent unstructured, the larger Tm(220-284(W269)) fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg(2+). NMR analysis shows that Mg(2+) induces chemical shift perturbations in both Tm(220-284(W269)) and Tm(259-284(W269)) in the vicinity of His276, in which are located several negatively charged residues.

Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach.

BACKGROUND: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma. OBJECTIVE: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides. METHODS: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program. RESULTS: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6. CONCLUSION: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum.

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.

Differential expression of tropomyosin during segmental heart development in Mexican axolotl.

The Mexican axolotl, Ambystoma mexicanum, serves as an intriguing model to investigate myofibril organization and heart development in vertebrates. The axolotl has a homozygous recessive cardiac lethal gene "c" which causes a failure of ventricular myofibril formation and contraction. However, the conus of the heart beats, and has organized myofibrils. Tropomyosin (TM), an essential component of the thin filament, has three known striated muscle isoforms (TPM1alpha, TPM1kappa, and TPM4alpha) in axolotl hearts. However, it is not known whether there are differential expression patterns of these tropomyosin isoforms in various segments of the heart. Also, it is not understood whether these isoforms contribute to myofibril formation in a segment-specific manner. In this study, we have utilized anti-sense oligonucleotides to separately knockdown post-transcriptional expression of TPM1alpha and TPM4alpha. We then evaluated the organization of myofibrils in the conus and ventricle of normal and cardiac mutant hearts using immunohistochemical techniques. We determined that the TPM1alpha isoform, a product of the TPM1 gene, was essential for myofibrillogenesis in the conus, whereas TPM4alpha, the striated muscle isoform of the TPM4 gene, was essential for myofibrillogenesis in the ventricle. Our results support the segmental theory of vertebrate heart development.

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin.

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.

Purification, characterization, and immunolocalization of paramyosin from the adult stage of Fasciola hepatica.

Paramyosin, a vaccine candidate in different helminthiases, was purified from the adult liver fluke Fasciola hepatica using two different procedures. The first started with a crude extraction of paramyosin in high-salt buffer followed by gel filtration chromatography and two precipitation-solubilization cycles; in the second, anion exchange chromatography replaced the gel filtration step. In both cases, the apparent molecular weight of the purified protein determined by sodium dodecyl sulfate gel electrophoresis under reducing and non-reducing conditions was 97 kDa and 200 kDa, respectively. The molecular weights were consistent with the presence of a dimeric protein linked by disulfide bridges. Western blot analysis showed that the dimeric and monomeric forms were both recognized by an antiserum raised against the F. hepatica 97 kDa band (alpha-FhPmy), and by an anti- Schistosoma mansoni paramyosin immune serum. Immunohistochemistry using alpha-FhPmy demonstrated the localization of paramyosin within the subtegumental muscle and in muscle cells surrounding the gut of adult parasites. We also observed labeling of extramuscular structures like testes, surface lamellae of the gut and the tegument of adult flukes.

A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization.

Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as alpha-amino acetylation of the N-terminus of the muscle protein. Nalpha-acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.

The calcium-induced switch in the troponin complex probed by fluorescent mutants of troponin I.

The Ca2+-induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the troponin complex.