The Functional Properties of Human Slow Skeletal Troponin T Isoforms in Cardiac Muscle Regulation

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, −exon 12), and HSSTnT3 (−exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (−exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca²⁺, respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca²⁺ sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca²⁺ sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.     

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component,Troponin T

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca²⁺ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca²⁺ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca²⁺ binding measurements. Ca²⁺ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca²⁺ sensitivity observed in Ca²⁺ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.     

胰岛素及cAMP对培养人动脉平滑肌细胞HDL受体功能的影响

对胰岛素ｃＡＭＰ对培养人动脉平滑肌细胞（ＳＭＣ）ＨＤＬ受体功能的影响进行了研究,结果发现：胰岛素使ＳＭＣＨＤＬ受体的结合容量Ｂｍａｘ即受体数目显著下降,而对ＳＭＣＨＤＬ受体的Ｋｄ值亲和力无影响;ｃＡＭｐ则ＳＭＣＨＤＬ受体亲和力增加,而对受体数目无影响。     

Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.     

Muscle-Specific Splicing of a Heterologous Exon Mediated by a Single Muscle-Specific Splicing Enhancer from the Cardiac Troponin T Gene

The chicken cardiac troponin T (cTNT) gene contains a single 30-nucleotide alternative exon that is included in embryonic striated muscle and skipped in the adult. Transient-transfection analysis of cTNT minigenes in muscle and fibroblast cell cultures previously identified four muscle-specific splicing enhancers (MSEs) that promote exon inclusion specifically in embryonic striated muscle cultures. Three MSEs located in the intron downstream from the alternative exon were sufficient for muscle-specific exon inclusion. In the present study, the boundaries of these MSEs were defined by scanning mutagenesis, allowing analysis of individual elements in gain-of-function experiments. Concatamers of MSE2 were necessary and sufficient to promote muscle-specific inclusion of a heterologous exon, indicating that it is a target for muscle-specific regulation. Sequences present in MSE2 are also found in MSE4, suggesting that these two MSEs act in a similar manner. MSE3 appears to be different from MSE2 and MSE4 yet is able to functionally replace both of these elements, demonstrating functional redundancy of elements that are likely to bind different factors. MSE2 and MSE4 each contain a novel sequence motif that is found adjacent to a number of alternative exons that undergo regulated splicing in striated muscle, suggesting a common role for this element in muscle-specific regulation.     

MappingTNNC1, the Gene That Encodes Cardiac Troponin I in the Human and the Mouse

We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7.     

A Positive GATA Element and a Negative Vitamin D Receptor-Like Element Control Atrial Chamber-Specific Expression of a Slow Myosin Heavy-Chain Gene during Cardiac Morphogenesis

We have used the slow myosin heavy chain (MyHC) 3 gene to study the molecular mechanisms that control atrial chamber-specific gene expression. Initially, slow MyHC 3 is uniformly expressed throughout the tubular heart of the quail embryo. As cardiac development proceeds, an anterior-posterior gradient of slow MyHC 3 expression develops, culminating in atrial chamber-restricted expression of this gene following chamberization. Two cis elements within the slow MyHC 3 gene promoter, a GATA-binding motif and a vitamin D receptor (VDR)-like binding motif, control chamber-specific expression. The GATA element of the slow MyHC 3 is sufficient for expression of a heterologous reporter gene in both atrial and ventricular cardiomyocytes, and expression of GATA-4, but not Nkx2-5 or myocyte enhancer factor 2C, activates reporter gene expression in fibroblasts. Equivalent levels of GATA-binding activity were found in extracts of atrial and ventricular cardiomyocytes from embryonic chamberized hearts. These observations suggest that GATA factors positively regulate slow MyHC 3 gene expression throughout the tubular heart and subsequently in the atria. In contrast, an inhibitory activity, operating through the VDR-like element, increased in ventricular cardiomyocytes during the transition of the heart from a tubular to a chambered structure. Overexpression of the VDR, acting via the VDR-like element, duplicates the inhibitory activity in ventricular but not in atrial cardiomyocytes. These data suggest that atrial chamber-specific expression of the slow MyHC 3 gene is achieved through the VDR-like inhibitory element in ventricular cardiomyocytes at the time distinct atrial and ventricular chambers form.     

Down Regulation of Genes Involved in T Cell Polarity and Motility during the Induction of Heart Allograft Tolerance by Allochimeric MHC I

Background

The allochimeric MHC class I molecule [α1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells.

Methodology/Principal Findings

To elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs).

Conclusions

These results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells'' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation.     

Identification of a Novel Human Gene Containing the Tetratricopeptide Repeat Domain from the Down Syndrome Region of Chromosome 21

The Down syndrome (DS) region on chromosome 21, which is responsiblefor the DS main features, has been defined by analysis of DSpatients with partial trisomy 21. Within the DS region, we constructeda 1.6-Mb P1 contig map previously. To isolate gene fragmentsfrom the 1.6-Mb region, we performed direct cDNA library screeningand exon trapping using the P1 clones and a human fetal braincDNA library, and obtained 67 cDNA fragments and 52 possibleexons. Among them, 23 cDNA fragments and 4 exons were interpretedto be derived from a single gene by localization on P1 clonesand by Northern analysis. To obtain the full-length cDNA sequence,longer cDNA clones were further screened from another humancDNA library which was enriched with longer cDNA species. Theseclones were sequenced and assembled to a sequence of 9045 bp.This transcribed sequence encodes a novel 2025 amino-acid proteincontaining tetratricopeptide repeat (TPR) motifs and thereforethe gene was designated as TPRD (a gene containing the TPR motifson the Down syndrome region). The TPR domain has been foundin a certain protein phosphatase and in other proteins involvedin the regulation of RNA synthesis or mitosis. The TPRD gene,the novel gene which was proved to be in the 1.6-Mb region andto have the interesting features described above, is a candidatefor genes responsible for the DS phenotypes.     

细胞角蛋白基因13在喉鳞状细胞癌中缺失和表达的研究

为了探讨细胞角蛋白基因13（Cytokeratin13,CK13)在喉癌发生中的作用,在CK13基因内部及附近选择5个微卫星引物进行杂合性丢失（loss of heterozygosity,LOH)分析,于DNA水平间接检测72例喉鳞状细胞癌患者中该基因的缺失,应用Northern Blot检测16例喉鳞癌患者的配对肿瘤及癌旁正常组织中CK13基因的表达差异;同时应用CK13蛋白单克隆抗体对不同分化程度的喉鳞癌组织进行免疫组化染色。结果表明：5个STR位点均存在LOH,其中D17S1964E、D17S2092、D17S791、D17S1665及D17S808位点的LOH频率分别为18．03％、28．13％、27．42％、39．68％和34．85％,其中D17S1665位点的LOH频率最高,至少一个位点出现LOH的病例高达77．78％（56／72）,杂合性丢失与临床分期、淋巴结转移无显著相关,但与肿瘤分化程度高低相关（P＜0．05）。Northern blot结果表明：16例喉鳞癌患者C13基因在正常组织中的表达比肿瘤组织显著增强,免疫组化结果也显示CK13蛋白在正常组织或高分化肿瘤中的表达明显高于低分化者,且存在显著差异（P＜0．01）。证实CK13基因可能在喉鳞状细胞癌的发生中具有重要作用,可能是一个新的抑癌基因。     

猪脂肪及肌肉组织中基因表达信息分析

为探测猪脂肪及肌肉组织中基因表达概况,利用猪EST资源和人类基因序列开展计算机模拟研究,旨在为猪肉质改良的遗传基础分析提供候选信息。执行Blast比对程序以识别人类基因组基因与猪EST序列间的同源性并筛选出高度同源记录,同时编制4个Java程序进行序列检索收集、序列比对结果的过滤筛选以及分类处理。统计分析表明：至少有2002个基因在猪脂肪及肌肉组织中表达,其中1087个基因在脂肪组织表达,1205个基因在肌肉组织中表达,两组织共同表达的基因为290个;筛选出高同源基因,同时分类统计出了114个基础活性基因(脂肪和肌肉组织分别表达80和34个),并选取Top记录进行了描述分析和总结。     

Electron Microscopic Studies of Skeletal and Cardiac Muscle of a Benthic Fish. I. Myofibrillar Structure in Resting and Contracted Muscle

SYNOPSIS. Electron microscopic studies are reported on glycerinatedskeletal and cardiac muscle of a benthic fish, Coryphaenoidesspecies. In white skeletal muscle, the sarcomeres have a restinglength of approximately 1.8 µ, with thick filaments 1.4µ and thin filaments 0.75 µ in length. These dimensionsare somewhat shorter than filament lengths of oilier vertebratemuscles, possibly due to the elfect of volume increase duringassembly of thick and thin filaments at high hydrostatic pressure.During ATP-induced contraction of Coryphaenoides muscle fromsarcomere lengths of 1.8 µ to 1.6 µ, there is acharacteristic interdigitation of thick and thin filaments,with decrease in I band length and no change in length of thickor thin filaments. However, in sarcomeres contracted to lengthsof 1.5 µ. to 1.2 µ, there is a slight shorteningof the A band, apparently due to shortening of thick filaments,that occurs despite the presence of residual I band in the samesarcomeres. There is no obvious crumpling or distortion of thickfilaments during contraction to sarcomere lengths as low as1.0 µ, but filament organization undergoes extensive disarrayat sarcomere lengths approaching 0.7 µ. Although effectsfrom heterogeneity of filament length cannot be excluded withcertainty, the present evidence does suggest that contractionot Coryphaenoides muscle from 1.6 µ to 1.0 µ sarcomerelengih is accompanied by shortening of thick filaments consequentto a structural change within the thick filament core.     

Prevalence of the Angiotensin I Converting Enzyme Gene Insertion/Deletion Polymorphism in a Healthy Turkish Population

Angiotensin converting enzyme (ACE) plays an essential role in the renin–angiotensin system. It converts angiotensin I to angiotensin II and inactivates bradykinin and tachykinins. Numerous studies have been published investigating associations of the ACE gene I/D polymorphism with various pathophysiological conditions. We examined the prevalence of the ACE I/D polymorphism in a sample of healthy volunteers from western Turkey, including 1063 healthy Turkish controls. Analysis of the ACE I/D gene polymorphisms by polymerase chain reaction found frequencies of 16.1% for the II genotype, 47.7% for the ID genotype, and 36.2% for the DD genotype. The allele frequency was 39.9% for the I alleles and 60.1% for the D allele. This study demonstrates that the allele and genotype frequency values for the Turkish population are similar to previously published frequencies for Caucasian populations.