Cell shape and cardiosphere differentiation: a revelation by proteomic profiling.

Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells) and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E) derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions.

Factors involved in signal transduction during vertebrate myogenesis.

Muscle is a contractile tissue of animals, dedicated to produce force and cause motion. In higher animals, there are two types of muscle tissue: (a) striated muscle, including all voluntary skeletal muscles and involuntary cardiac muscle, and (b) smooth muscle consisting of involuntary muscles, including those of the viscera, blood vessels, and uterus. Although muscle growth and regeneration take place throughout vertebrate life, the heart is the first organ to start functioning, with continued development until delivery. Skeletal muscles, on the other hand, develop in four successive, temporally distinct phases of embryonic, fetal, neonatal, and adult muscle with the postnatal phase being basically hypertrophy. Unlike terminally differentiated skeletal and cardiac muscles in adults, smooth muscle cells retain their plasticity and the phenotype can change reversibly in response to environmental changes. For the past 20 years, the availability of gene recombination technology directed the focus of studies on transcription factors and signaling molecules, and we would like to review what has been explored by recent studies on myogenesis.

Proteomic comparison of spherical aggregates and adherent cells of cardiac stem cells.

BACKGROUND: Previously published papers showed that cardiac stem cells (CSCs) form (cardio)sphere. However, recent studies questioned the significance of the sphere-formation as one of the characteristics of CSCs. We isolated c-kit-positive cardiac stem cells, cultured as bulk (CSC-BC) and characterized them previously. Among them, CSC-BC21 formed an extraordinary number of spheres. Using a clone derived from this bulk culture, we investigated the effect of sphere-formation on differentiation and performed proteomics analysis comparing two statuses, cardiosphere and dish substrate attachment. METHODS: We performed sphere-forming assay to compare the sphere-forming ability among CSC-BCs. The cloned cells from CSC-BC21, which had distinct sphere-forming ability, were cultured in a differentiation medium (DM) to induce cardiac myocyte differentiation. We performed RT-PCR analysis to investigate if cardiosphere-formation affects cardiac myocyte gene expression level. Furthermore, proteome analysis was performed to compare floating cardiosphere (flCS) and dish-attached cardiosphere-derived cells (daCS). RESULTS: One of the cloned cells, CSC-21E expressed higher troponin I message than CSC-BC21. Moreover, the message level of troponin I was enhanced when they had experienced cardiosphere prior to the treatment of myocyte differentiation medium. The change from flCS to daCS accompanied up-regulation of chaperones and down regulation of glycolytic and other metabolic enzymes. Calreticulin and Hsp 90 were among the up-regulated chaperons. Calreticulin is known to be an essential component of cardiogenesis. CONCLUSION: These results suggest that the switch from aggregated sphere to the cell attachment, is important for advancing the cardiac cell differentiation.

Genomic sequence encoding diversity segments of the pig TCR delta chain gene demonstrates productivity of highly diversified repertoire.

To better understand the function and diversity of gammadelta T cells, we determined the genomic sequence encoding diversity (D) segments of the porcine TCR delta chain and its upstream regions, because pigs and other artiodactyls have relatively high proportions of gammadelta T cells. The revealed sequence contained 28 variable (V) alpha/delta segments, including 4 TRDV1 and at least 6 Ddelta segments, a much higher number than in humans and mice. All 6 of the Ddelta segments that had canonical recombination signal sequences were functionally utilized in expressed TCR delta chain genes. The multiplicity of Ddelta segments enabled the use of more than 3 Ddelta segments in a single functional TCR delta chain. The increased number of TCR delta segments was acquired by the duplication of the germline sequence, which occurred after the divergence of artiodactyls from primates and rodents. These data demonstrate that the pig is able to generate a highly diversified repertoire of TCR delta chain molecules.

Porcine T-cell receptor beta-chain: a genomic sequence covering Dbeta1.1 to Cbeta2 gene segments and the diversity of cDNA expressed in piglets including novel alternative splicing products.

Porcine TCRbeta-chain cDNA clones were isolated from thymic and peripheral blood lymphocytes of piglets. Using these nucleotide sequences, a genomic 18kbp sequence stretch covering Dbeta1 to Cbeta2 gene segments was identified, which revealed that the porcine TCRbeta-chain locus consists of two sets of Dbeta-Jbeta-Cbeta gene groups with each set having a Dbeta gene segment, seven Jbeta gene segments and a down stream Cbeta gene segment composed of four exons. This structure is consistent with other known mammalian TCRbeta-chain loci. With this genomic information, TCRbeta-chain clones from cDNA libraries were analyzed. Sixteen Vbeta gene segments were obtained accompanied by either Dbeta1 or Dbeta2 and by one of the nine Jbeta gene segments. Five different Cbeta cDNA sequences were obtained including four types of Cbeta1 sequences and one type of Cbeta2 sequence. The differences among the Cbeta1 sequences are either allelic polymorphisms or two splice variants, one being a product of exon1 splicing to exon3 (exon2 skipping), and another being an alternative splicing using a splice acceptor site newly discovered inside Cbeta1 exon4. The latter splice acceptor site was also found in human, mouse and horse all giving short cytoplasmic domain with Phe at their C-terminal ends. Other splicing products included trans-splicing of Jbeta2 to Cbeta1, non-functional splicing of two Jbeta gene segments in tandem and a part of Jbeta2.7-Cbeta2 intron to Cbeta2 exon1. Numerous examples of splice variants may suggest the involvement of splicing in generating TCRbeta-chain functional diversity.

The genomic structure and a novel alternatively spliced form of porcine pTalpha chain.

A complete genomic nucleotide sequence for porcine pTalpha gene was obtained from a BAC clone, which revealed a novel exon 2 missing in human and murine counterparts. Cattle and dog genomic sequences showed the counterparts corresponding to porcine exon 2. Using thymocyte RNA and RT-PCR, three types of porcine pTalpha-chain cDNA sequences, pTalpha1, pTalpha2 and pTalpha3, were obtained. These three different cDNA sequences were alternatively spliced products with pTalpha1 consisting of exons 1, 2, 3, 4, and 5, pTalpha2 consisting of exons 1, 2, 4, and 5, and pTalpha3 consisting of exons 1, 2, 3 and the intron down stream of exon 3. pTalpha1 and pTalpha2 correspond to previously reported pTalphaa, and pTalphab, respectively, and pTalpha3 is reported for the first time. Using RT-PCR, pTalpha3 appeared expressed predominantly in the thymocyte RNA. The chromosome location of pTalpha was investigated using Radiation Hybrid Map and FISH, both of which revealed the location at SSC7q11-q12.

Jalpha-gene segment usage and the CDR3 diversity of porcine TCRalpha-chain cDNA clones from the PBL of a five-month-old pig and the thymus of a one-month-old pig.

Porcine T-cell receptor alpha (TCRalpha)-chain cDNA clones were isolated from libraries made from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes (PBL) of a 5-month-old Clawn strain pig. Among 109 cDNA clones with the Jalpha-gene segment, 44 different Jalpha-gene segments were found out of the 61 Jalpha-gene segments previously identified in the porcine germline sequence. Among the 103 complete TCRalpha-chain cDNA clones with the rearranged Valpha- and Jalpha-gene segments, 33 different Valpha-gene segments were identified, which randomly rearranged to Jalpha-gene segments indicating lack of any specific combinations between Valpha- and Jalpha-gene segments with only one exception of the same set of Jalpha-gene segments in duplicate clones. Among the cDNA clones from PBL of an individual 5-month-old Clawn strain pig, a broad distribution of the Jalpha-gene segment usage was observed over the entire Jalpha-gene cluster. The Jalpha-gene segment usage in an individual 1-month-old thymus from a LW strain pig also gave a pattern consistent with the 5-month-old pig. These distributions of the Jalpha-gene segment usage were similar to the previously reported patterns for human T-cells and those of adult murine T-cells. Among the porcine cDNA clones isolated, TCRalpha-chain CDR3 length ranged from 4 to 14 amino acids with the average being 9.35 amino acids. Present report provides groundwork for further studies on porcine TCRalpha-chain expression.

Porcine TCR CD3zeta-chain and eta-chain.

Complete porcine CD3zeta-chain cDNA sequence was obtained for the first time, and its genomic nucleotide sequence was investigated from exon 2 down to CD3eta-chain exon 8. The sequence of porcine CD3zeta-chain showed homologous amino acid sequence with human and murine counterparts, in contrast to CD3eta-chain exon 8 with diversity among animals previously investigated. CD3eta-chain peptide is an alternative splice form of CD3zeta-chain exon 7 splicing to CD3eta-chain exon 8 instead of CD3zeta-chain exon 8. The genomic sequences revealed that the splice acceptor sequences of CD3eta-chain exon 8 of all animals investigated to be completely uniform. Further, CD3eta-chain exon 8 amino acid sequences retained the unique characters of having high proline (Pro) and positively charged amino acid content except for rats and mice. Although the biological role of CD3eta-chain remains to be enigmatic, these evidences suggests the evolutional pressure to maintain its sequence.

TRAV gene usage in pig T-cell receptor alpha cDNA.

Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.

Involvement of IRAK-M in peptidoglycan-induced tolerance in macrophages.

The molecular mechanisms by which pathogen-associated molecular patterns recognized by TLR2, such as peptidoglycan (PGN), induce homotolerance are largely unknown. It was recently reported that IRAK-M negatively regulates TLR signaling. In this study, we elucidate the molecular mechanisms of tolerance induced by PGN, with a focus on the role of IRAK-M. We demonstrate that pretreatment of macrophage RAW264.7 cells with a high concentration (30 microg/ml) of PGN for 16 h effectively induces tolerance against following stimulation with 30 microg/ml of PGN; while pretreatment with a low concentration (1 microg/ml) of PGN does not. IRAK-M is induced in cells treated with the high concentration of PGN 4-24 h after PGN stimulation, but not in cells treated with the low concentration of PGN up to 24 h after stimulation. Phosphorylation of MAPKs and IkappaBalpha is inhibited after the second PGN stimulation in tolerant cells. Kinase activity of IRAK-1 and association between IRAK-1 and MyD88 are also suppressed in PGN-induced tolerant cells. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates the production of TNF-alpha after PGN restimulation. These results suggest that induction of IRAK-M and inhibition of kinase activity of IRAK-1 are crucial to PGN-induced tolerance in macrophages.

Genomic structure around joining segments and constant regions of swine T-cell receptor alpha/delta (TRA/TRD) locus.

A complete genomic region of 131.2 kb including the swine T-cell receptor alpha/delta constant region (TRAC/TRDC) and joining segments (TRAJ/TRDJ) was sequenced. The structure of this region was strikingly conserved in comparison to that of human or mouse. All of the 61 TRAJ segments detected in the human genomic sequence were detected in the swine sequence and the sequence of the protein binding site of T early alpha, the sequence of the alpha enhancer element and the conserved sequence block between TRAJ3 and TRAJ4 are highly conserved. Insertion of the repetitive sequences that interspersed after the differentiation of the species in mammals such as short interspersed nucleotide elements is markedly suppressed in comparison to other genomic regions, while the composition of the mammalian-wide interspersed sequences is relatively conserved in human and swine. This observation indicates the existence of a highly selective pressure to conserve this genomic region around TRAJ throughout the evolution of mammals.

Myocarditogenic epitopes and autoimmune myocarditis.

Experimental autoimmune myocarditis is provoked by immunization with cardiac myosin. This animal model finally develops into dilated cardiomyopathy through repetitive myosin injections. To identify the myocardiogenic epitope, therefore, it is imperative not only to understand the mechanism of induction, but also to produce specific therapies, such as a blocking therapy to suppress the autoimmune process. Thus, we attempted to identify the myocarditogenic epitope using recombinant peptides. Beta-cardiac myosin heavy chain (CMHC) was amplified from rat mRNA by a reverse transcription polymerase chain reaction method. The PCR primers were designed to narrow the epitopic amino acid portion from each N-terminal to C-terminal site. These PCR products were cloned into an E. coli expression vector to produce fusion proteins consisting of a Histidine-tag and a myosin peptide. The segment of amplified CMHC including the epitopic amino acid sequence to provoke moderate myocarditis in vivo was reported previously. Each peptide solution was emulsified in an equal volume of complete Freund's adjuvant and given as an immunization to 7-week-old rats. On day 21 after immunization, the rats were sacrificed, and the fresh heart was observed pathologically. Through this immunization, we could restrict the myocardiogenic site. Lastly, this peptide was found to be located on residues from 1,124 to 1,153. Using ELISA, the antibodies against myocarditogenic peptides were easily identified. Whether or not the antibody productivity is linked to myocarditogenecity is discussed.

DNA methylation dominates transcriptional silencing of Pax5 in terminally differentiated B cell lines.

Pax5 plays a key role in the progression of B cell development. Its expression is observed in a wide range of cell types from early lineage-committed precursors up to mature B cells, but is silenced in terminal differentiated plasma cells. In this report, we show that DNA methylation is involved in the silencing of Pax5. In the Pax5-expressing cell lines 38B9 (pre-B) and 2PK-3 (mature B), all CpG sites in TATA-containing upstream promoter were unmethylated, whereas these sites were completely methylated in myeloma cell lines FO and Sp-2/0, which do not express Pax5. Demethylation of FO and Sp-2/0 with 5-aza-2'-deoxycytidine (5-aza-dC) resulted in Pax5 re-expression with the concomitant expression of CD19 and mb-1 genes, which are known to be the target genes of Pax5. Re-expression of Pax5 was also induced by trichostatin A (TSA), which was a specific inhibitor of histone deacetylase. This re-expression was, however, transcribed only from the TATA-less downstream promoter. Taken together, we concluded that the upstream promoter was predominantly inactivated by DNA methylation, while the downstream promoter was repressed by the histone deacetylation. This synergetic inactivation of two promoters results in the final silencing of Pax5 expression in terminally differentiated B cell lines.

A mouse with a point mutation in plasma membrane Ca2+-ATPase isoform 2 gene showed the reduced Ca2+ influx in cerebellar neurons.

We analyzed mutant mice showing behavioral defects such as severe tremor, up-and-down and side-to-side wriggling of neck without coordination, and found that the gene causing the defects was located between 46 and 60.55 centimorgans (cM) on the mouse chromosome 6. In this region, nucleotide transition of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene was found, which caused a glutamic acid to change into lysine. Since PMCA2 is expressed in the cerebellum and plays an important role to maintain the homeostasis of the intracellular Ca2+ as a Ca2+ pump, the behavioral defect can be ascribed to the impairment of Ca2+ regulation in neurons of the cerebellum. To confirm the defect of Ca2+ homeostasis in the mutant mice, we measured high K+-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in the cerebellar neurons. Contrary to our expectation, the extent of the [Ca2+]i increase in all the regions tested in the cerebellar slice was far smaller than that of the wild type mice, while the resting [Ca2+]i remained almost unaltered. The rate of rise in [Ca2+]i during high K+-induced depolarization was significantly reduced, and the extrusion rate of increased [Ca2+]i was also reduced. These results suggested that voltage-gated Ca2+ channels were down-regulated in the mutant mice in order to regulate [Ca2+]i toward the normal homeostasis. The behavioral defects may be ascribed to the down-regulated Ca2+ homeostasis since dynamic changes in [Ca2+]i are important for various neuronal functions.

Porcine Fas-ligand gene: genomic sequence analysis and comparison with human gene.

Thymic Fas-ligand (FasL) cDNA and hepatic FasL genomic sequences were obtained from a 2-month-old LW pig. From these nucleotide sequences, amino acid sequence was deduced and compared with FasL sequences obtained from various animals. This comparison reveals that porcine FasL is closer to that of human, macaca and cat, and differs more from mouse and rat. The extracelluar domains of porcine and human FasL proteins appear to be functionally compatible. The complete genomic DNA sequence of porcine FasL was also compared with its human counterpart. Exons showed 80-89% nucleotide homology between pig and human, while introns showed 64-69% nucleotide homology. Sequence comparison by Harr plot analysis revealed many stretches within introns having identical sequences, suggesting that the sites may have unidentified common functions. One potential extra exon between exons 2 and 3 was located within porcine intron 2. This potential exon has no counterpart in human FasL intron 2. Whether or not this extra exon can be expressed and could cause additional immunological responses remains to be investigated. For future xenotransplantation, it is important to compare porcine and human genomic sequences, and to investigate their system compatibilities.