Decoding the Reproductive System of the Olive Fruit Fly, Bactrocera oleae.

In most diploid organisms, mating is a prerequisite for reproduction and, thus, critical to the maintenance of their population and the perpetuation of the species. Besides the importance of understanding the fundamentals of reproduction, targeting the reproductive success of a pest insect is also a promising method for its control, as a possible manipulation of the reproductive system could affect its destructive activity. Here, we used an integrated approach for the elucidation of the reproductive system and mating procedures of the olive fruit fly, Bactrocera oleae. Initially, we performed a RNAseq analysis in reproductive tissues of virgin and mated insects. A comparison of the transcriptomes resulted in the identification of genes that are differentially expressed after mating. Functional annotation of the genes showed an alteration in the metabolic, catalytic, and cellular processes after mating. Moreover, a functional analysis through RNAi silencing of two differentially expressed genes, yellow-g and troponin C, resulted in a significantly reduced oviposition rate. This study provided a foundation for future investigations into the olive fruit fly's reproductive biology to the development of new exploitable tools for its control.

A Mechanism Underlying Sex-Associated Differences in Ankylosing Spondylitis: Troponin C2, Fast Skeletal Type (TNNC2) and Calcium Signaling Pathway.

BACKGROUND Ankylosing spondylitis (AS) is a disease that causes pathological changes in the spine and sacroiliac joints. Numerous studies have shown that the characteristics of AS differ between males and females. The purpose of this study was to discover the key molecules that contribute to sex-associated differences in AS, which may provide a new molecular target for personalized treatment. MATERIAL AND METHODS The gene expression profile of GSE39340 was downloaded from the Gene Expression Comprehensive database, and 2 groups (AS vs. No-AS groups and male AS vs. female AS groups) of differentially expressed genes (EDGs) were obtained by GEO2R. The DAVID database was used for DEGs function and enrichment analysis. Based on data in the STRING online database, a protein-protein interaction (PPI) network was constructed in Cytoscape. Hub genes were selected from CytoHubba. With the intersection of the top 30 hub genes of 2 sets of EDGs, genes coexisting with the KEGG-related pathway were found. RESULTS We screened 560 genes between the AS and No-AS groups, and screened 710 genes that were differentially expressed between the male and female AS groups. GO analysis showed that DEGs were mainly co-enriched in molecular functions, including structural constituent of muscle. The KEGG pathway mainly included the structural constituent of muscle. Seven hub genes were obtained. Troponin C2 and fast skeletal type (TNNC2) were the key genes participating in the calcium signaling pathway. CONCLUSIONS This study contributes to understanding the molecular biological mechanism underlying sex-associated differences in AS. TNNC2 and calcium signaling pathway may be new targets for the individualized treatment of AS.

TNNC1 Reduced Gemcitabine Sensitivity of Nonsmall-Cell Lung Cancer by Increasing Autophagy.

BACKGROUND As we know, chemotherapy resistance is a critical factor leading to recurrence and metastasis of nonsmall-cell lung cancer (NSCLC). To clarify the key target and potential mechanism of resistance to gemcitabine (GEM) in NSCLC, we selected Gene Expression Omnibus Data Set and statistically analyzed a parent cell group and a GEM-resistant cell group. Results showed that the expression of troponin C1, slow skeletal and cardiac type (TNNC1) in GEM-resistant cells was higher than in parent cells, which implies that TNNC1 was associated with GEM resistance in lung cancer cells. MATERIAL AND METHODS TNNC1 expression level was detected by reverse transcription-quantitative polymerase chain reaction or western blot in GEM-resistant patient serum and cell lines. It could reduce or increase autophagy response and GEM resistance accordingly by inhibition of the short interfering ribonucleic acid or by forced overexpression of TNNC1 viruses in A549 cell line and GEM-resistant cell line (A549/GemR) respectively. Blocking autophagy with 3-methyladenine increased the sensitivity of chemotherapy confirmed by flow cytometry and microtubule-associated protein 1A/1B - light chain 3 punctate assay. What's more, in a loss-of-function model, silencing of forkhead box 03 (FOXO3) in A549/GemR cells could rescue the autophagy weakened by TNNC1. RESULTS TNNC1 promoted GEM chemoresistance of NSCLC by activating cytoprotective autophagy, regulated negatively by FOXO3. This research may provide a completely new strategy for NSCLC treatment. CONCLUSIONS Targeting the TNNC1/FOXO3 signaling pathway in NSCLC may be a novel strategy to combat GEM resistance.

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag.

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner.

[Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²âº Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

Herpesvirus-mediated delivery of a genetically encoded fluorescent Ca(2+) sensor to canine cardiomyocytes.

We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca(2+) sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (I(to)), kinetics of the intracellular Ca(2+) transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the I(to) current was significantly changed and no major shifts occurred in parameters of [Ca(2+)](i) transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression.

Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca(2+) regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in Escherichia coli is an essential approach to preparing human TnI and mutants for structural and functional studies. The expression level of cardiac TnI in E. coli is very low. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow troponin C (TnC), a natural binding partner of TnI and a protein that readily expresses in E. coli at high levels. The co-expression moderately increased the expression of cardiac TnI although a high amount of TnC protein was produced from the bi-cistronic mRNA. The use of an E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons improved the expression of cardiac TnI. Modifications of two 5'-regional codons that have predicted low usages in bacterial cells did not reproduce the improvement, indicating that not the 5' but the overall codon usage restricts the translational efficiency of cardiac TnI mRNA in E. coli. However, deletion of the cardiac TnI-specific N-terminal 28 amino acids significantly improved the protein expression independent of the host cell tRNA modifications. The results suggest that the regulatory N-terminal domain of cardiac TnI is a dominant factor for the incompatibility in bacterial cells, supporting its role in modulating the overall molecular conformation.

Bla g 6: a troponin C allergen from Blattella germanica with IgE binding calcium dependence.

BACKGROUND: The known cockroach allergens do not appear to account for the full repertoire of IgE responses. OBJECTIVE: To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy. METHODS: A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. RESULTS: Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens. CONCLUSION: Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. CLINICAL IMPLICATIONS: Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.

Transient expression of fast troponin C transcripts in embryonic quail heart.

Most myofibrillar proteins, including troponin I and troponin T subunits of troponin complex, undergo developmental stage-specific isoform transitions in vertebrate heart before attaining adult contractile and regulatory characteristics. Only the cardiac/slow skeletal muscle type isoform of troponin C, however, has been shown to be expressed in both adult and developing heart. The changes in troponin C could be functionally important as the TnC isoforms vary in their affinities for Ca(2+). For example, fast troponin C has two Ca(2+) binding sites while slow/cardiac troponin C has a single regulatory site. This study demonstrates the co-expression of both fast and slow transcripts of troponin C in not only quail embryonic skeletal muscle but also embryonic heart using two different analytical techniques of polymerase chain reaction and in situ hybridisation procedure. Fast troponin C expression in the quail heart using in situ hybridisation procedure was first observed at embryonic day 3, with maximum expression at day 5 after which its level in the developing heart was gradually down regulated. In situ hybridisation staining of sections at these developmental stages demonstrated the expression of both fast and slow transcripts of troponin C in all cardiomyocytes.

Overexpression of sTnC polypeptide in muscle cells is controlled by its rapid degradation.

The check-points that maintain stoichiometric synthesis of muscle proteins were examined by misexpression of slow troponin C (sTnC) in mouse C2 myotubes. The sTnC mRNA was overexpressed in myotubes by transfecting these cells with a plasmid construct containing the constitutive CMV promoter-driven sTnC cDNA. An approximately four-fold increase of sTnC mRNA level in the transfected cells was observed. However, the increased mRNA level did not produce a corresponding increase of the sTnC polypeptide level in transfected cells. Only a modest 1.5-fold increase of the sTnC polypeptide level in the transfected cells was observed. The excess sTnC polypeptide in transfected cells was found in the soluble form which was not complexed with other thin filament proteins. The difference between the increase of sTnC mRNA and the polypeptide levels in transfected cells was not due to inefficient translation of the overexpressed sTnC mRNA. Analyses of the stability of the sTnC polypeptide in the thin filament and in the unassembled soluble forms showed that the excess soluble sTnC polypeptide was degraded more rapidly than the sTnC polypeptide of the thin filament. Analyses of the mRNA and polypeptide levels of several thin filament complements showed no effect of overexpression of the sTnC mRNA.

Alterations in contractile properties and expression of myofibrillar proteins in wobbler mouse muscles.

The purpose of this study was to characterize the alterations in muscle contractile (tension-pCa relationship) and biochemical (myosin heavy and light chains, troponin C content) properties in a hereditary motoneuron disease. The study was performed on wobbler mouse mutants which presented a neuronal degeneration. The time course of the disease was followed at 5 and 7 weeks in sternocleidomastoid (SCM) and soleus muscles. The wobbler disease was found to induce a shift from fast to slow myosin heavy-chain isoform expression in SCM and soleus muscles. The analysis of the myosin light-chain (MLC) composition revealed, for the SCM muscles, the appearance of the slow isoforms at 5 weeks and an increase in the regulatory MLC2 content at 7 weeks. A significant increase in the slow troponin C isoform content was found in both types of wobbler muscles at 7 weeks. The wobbler soleus and SCM muscles presented an age- and fiber-type-related atrophy, characterized by a decline in absolute maximal tension and fiber diameter. A decrease in calcium sensitivity was observed at 7 weeks for the soleus fibers and at both 5 and 7 weeks for the SCM. The results indicated fast-to-slow changes in contractile and biochemical properties of the wobbler soleus and SCM muscles, which occurred during the motoneuron degeneration process previously described in the wobbler pathology.

Delayed embryonic development of mouse masseter muscle correlates with delayed MyoD family expression.

While the masseter muscle is known to have several unique developmental characteristics as compared with other skeletal muscles, little is known about its myogenesis. Thus, we examined the expression of myogenic marker and of myoD family gene mRNA from embryonic day (E) 11 to birth. The obtained results were compared with our earlier results of the mouse tongue muscle, which is also involved in oral functions. The mRNA quantities were determined by means of the reverse-transcription and competitive-polymerase chain-reaction techniques. The expression of myogenic marker mRNA indicated that differentiation and maturation in the masseter began at E13 as in the tongue, and were not yet completed at birth, although they were completed in the tongue. The expression of myoD, myogenin, and myf5 mRNA peaked later in the masseter (E17) than in the tongue (E13). The expression of MRF4 mRNA began later in the masseter (E15) than in the tongue (E13). These results suggest that the delayed expression of the myoD family genes in the masseter correlates with delayed differentiation and maturation, probably due to the later functional requirements of the masseter than of the tongue.

Investigation of a genetically engineered mutant of barnacle troponin C containing a central helix deletion.

To examine the importance of the central alpha-helix of troponin C (TnC) we have bacterially expressed one of the isoforms of barnacle TnC (BTnC2), BTnCWT, but without the aspartate residue at position 80 in the central helix (BTnC80-). This manipulation is expected to produce an approximately 100 degrees axial rotation of the C-domain with respect to the N-domain, and a net charge change of -1. BTnC80- mutant was able to restore force to TnC-depleted skinned barnacle myofibrillar bundles to a greater extent than wild-type protein (approximately = 170%). Competition experiments between BTnC80- and BTnC2-4-, a mutant lacking both of the calcium-specific sites (sites II and IV), shows that deletion of a single amino acid in the central helix results in a protein with increased affinity for the thin filament and one that is bound preferentially compared to BTnC2-4- when at equimolar concentrations.

Stabilization of slow troponin C polypeptide compensates for its reduced synthesis in antisense oligodeoxynucleotide-treated cells.

The expression of genes for contractile proteins during myogenesis is coordinately regulated. Uncoupling the expression of the slow/cardiac troponin C (sTnC) gene from this process with an antisense phosphorothioate oligodeoxynucleotide (ODN) was used to examine the presence of any post-transcriptional mechanisms for regulating muscle protein synthesis. Approximately 70 and 50% decreases in sTnC polypeptide synthesis and mRNA levels, respectively, were achieved after 4 days antisense treatment. This decrease in sTnC polypeptide synthesis was not reflected in a similar decline in the steady-state level of this polypeptide. Extension of the ODN treatment to 7 days was required to produce a substantial decrease in the steady-state level of sTnC polypeptide. Our investigation suggests that during the 4-day treatment, the affected cells stabilized the sTnC polypeptide level by increasing its half-life. However, the stabilizing effect appears to be overridden during prolonged (7 days) antisense ODN treatment. Measurement of the polypeptide synthesis and mRNA levels of several contractile proteins showed no evidence of cross-regulation among the genes to coordinately regulate their expression levels.

Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.

The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.

Inhibition of troponin C production without affecting other muscle protein synthesis by the antisense oligodeoxynucleotide.

The effect of blocking expression of a specific gene with antisense phosphodiester oligodeoxynucleotides on the coordinate regulation of myogenesis was studied. Different regions of both fast and slow troponin C (TnC) mRNAs were targeted for binding of the antisense oligomer. The 5'-cap region of both mRNAs was found to be the most effective target for inhibiting the expression of these genes. Approximately 40%-60% inhibition of expression of a specific isoform of TnC was achieved. However, inhibition of the TnC expression did not appreciably alter the pattern of myogenesis of mouse C2C12 cells. The differentiated murine muscle cells were able to cope with this reduced level of the target gene expression by antisense phosphodiester oligomers. We have also used a phosphorothioate oligomer targeted against a common sequence within the coding region of both fast and slow TnC mRNAs. This oligomer was found to be ineffective in blocking TnC gene expression.

Fine mapping of five human skeletal muscle genes: alpha-tropomyosin, beta-tropomyosin, troponin-I slow-twitch, troponin-I fast-twitch, and troponin-C fast.

In this paper the chromosomal localization of the human skeletal muscle genes Troponin-I slow-twitch (TNNI1), Troponin-I fast-twitch (TNNI2), and Troponin-C fast (TNNC2) and the refinement of the position for alpha-Tropomyosin (TPM1) and beta-Tropomyosin (TPM2) are reported. By radiation hybrid mapping, TPM1 was assigned to chromosome 15q22.1, TPM2 to chromosome 9p13.2-p13.1, TNNI1 to chromosome 1q31.3, TNNI2 to chromosome 11p15.5, and TNNC2 to chromosome 20q12-q13.11. The genomic distribution of these genes is discussed, with particular emphasis on the cluster organization of the Troponin genes.

Slow troponin C gene expression in chicken heart and liver is regulated by similar enhancers.

Two isoforms of troponin C (TnC) are encoded by distinct single copy genes. Expression of fast TnC is restricted to the skeletal muscle, whereas the slow isoform is expressed in both skeletal and cardiac muscle. Chicken slow TnC (cTnC) gene is also expressed in some non-muscle tissues like the liver and the brain. Expression of cTnC gene is regulated by two distinct enhancers in cardiac and skeletal muscles. The cardiac specific enhancer is located in the immediate 5' flanking region (bp-124 to -79) of the murine cTnC gene whereas the skeletal enhancer is located within the first intron (bp 997 to 1141). In the present study we have examined how cTnC gene expression is regulated in the chicken liver. Transient transfection of liver cells with CTnC-CAT reporter constructs containing various regions of the murine cTnC gene showed that its expression in chicken liver is regulated by the cardiac specific enhancer. Furthermore, electrophoretic mobility shift assays using synthetic oligonucleotides corresponding to both CEF-1 and CEF-2 regions of the murine cardiac enhancer revealed formation of specific DNA-protein complexes. Ultraviolet light induced covalent linking of nuclear proteins to CEF-1 and CEF-2 oligomers were used to examine the nature of the cardiac enhancer binding polypeptides; one polypeptide of 48 kDa appeared to bind to both CEF-1 and CEF-2 sequences.