Biomedical subjects
Publications and source records attributed to S Tajima.
The temporoparietal fascial flap is useful in reconstructions of the hands and feet as tendon gliding material, but it is difficult to monitor its viability postoperatively. We have monitored such flaps by attaching a small preauricular skin flap 2 x 1 cm in size to the vascular pedicle. This preauricular island flap can be used as a "monitoring" flap.
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We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent demethylating reagent. The amount of transcript increased with 5-azacytidine treatment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107,254 Da. Specific antibodies raised against the fusion protein including glutathione S-transferase revealed an approximately 130-kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.
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Familial hypercholesterolemia is a genetic disorder caused by mutations of the low-density-lipoprotein (LDL) receptor gene. We characterized the structures of LDL receptor mRNA transcripts in the fibroblasts of a homozygous patient carrying a single base substitution (T-->C) at the 5' splice donor site of intron 12 of the LDL receptor gene. We identified three aberrant transcripts as a consequence of intron-12 read-through, exon-12 skipping and utilization of a cryptic splice donor site. Only a point mutation at the 5' splice donor site caused the production of three alternatively spliced products. None of these transcripts produced a functional LDL receptor protein in this patient.
Synthetic elastin peptides, VPGVG or its polymer (VPGVG)n, enhanced the proliferation of smooth muscle cells 1.5-fold during 48 h treatment at the concentrations over 10(-6) M or 1.0 microgram/ml, respectively. Monomeric and polymeric VPGVG sequences reduced elastin synthesis and its mRNA level to one-third and one-half of control respectively under the conditions in which the proliferation of cells were enhanced, but did not change collagen synthesis as measured by bacterial collagenase digestion. The elastin-specific autoregulation by elastin fragments may reflect the feedback regulation of elastin expression which may play an essential role in elastin metabolism under the normal and diseased conditions.
A study was made of the relationship between elastin expression and the proliferative state of chick vascular smooth-muscle cells. Confluent cells of primary culture brought to a quiescent state by the deprivation of serum for 72 h exhibited a 5-, 3.5- and 2-fold increase in elastin synthesis, elastin mRNA level and transcriptional activity of elastin gene respectively over those in the proliferative state. On re-addition of serum in serum-deprived culture, cells started to proliferate, and elastin synthesis, its mRNA level and transcription of the gene decreased to the level of a proliferative state within 24 h, indicating that elastin expression in smooth-muscle cells was controlled by their growth states at least in part at a transcriptional level. A comparable increase in elastin mRNA level was observed when the cell growth was arrested by suspension culture for 72 h. When the cells were synchronized at the G1/S phase with thymidine/hydroxyurea treatment, elastin expression at the G1/S phase was greater than that at the G2/M phase during cell cycling. Elastin mRNA level at the G0 phase brought about by serum-deprivation or suspension culture predominated over that at the G1/S phase during cell cycling. These results indicate that gene expression of elastin and cell cycle are tightly coupled, which is independent of the presence of serum or adhesive state, and that elastin expression could be a biochemical marker for the growth states of smooth-muscle cells.
We overexpressed mouse DNA methyltransferase in murine C2C12 myoblast cells and tested the isolated clones for their ability to differentiate. Significant numbers of the clones showed distinct myotubes 24 h after the isolated transformants had been induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point. Transfection of the vacant vector or the plasmid containing the reverse-oriented DNA methyltransferase cDNA did not provide significant numbers of transformants with the accelerated differentiation phenotype, suggesting that the effect is caused by the expression of DNA methyltransferase. The expressions of skeletal muscle myosin and creatine kinase in clones that showed the accelerated differentiation-phenotype were also induced about 24 h earlier and at higher levels relative to the parent C2C12 or the control cells, indicating that the entire process of myogenesis had been accelerated. All the methyltransferase-transfected clones, regardless of their phenotypes, demonstrated about threefold higher DNA methyltransferase activity and higher methylation levels than those of the clones transfected with vector alone or the reverse-oriented plasmid. At the early stage of transfection of the sense-oriented plasmid, high de novo methylation activities were detected. We consider it likely that this high de novo methylation activity is the reason for the high methylation levels and the accelerated myotube formation of the clones transfected with the sense-oriented plasmid. In some transformants which showed the accelerated differentiation phenotype, MyoD1 was already fully expressed under the growth conditions while, in control cells, MyoD1 was expressed at low levels. This elevated level of MyoD1 transcription could account for the accelerated myotube formation observed in the transformants. The methylation state of the HpaII sites in exon 1 through exon 2 of the MyoD1 gene and the expression of the MyoD1 transcript are positively correlated.
cDNA clones encoding the bovine major histocompatibility complex (MHC) class II DQ alpha chain were isolated. One clone, MQ9, encoded a primary translated product of 255 amino acids, with a signal peptide of 23 amino acids and a mature polypeptide of 232 amino acids. A new A2 gene in the DQ subregion of the bovine genome was identified from a comparison of amino acid sequences encoded by class II A genes among several species and the construction of a phylogenetic tree. It was revealed that MQ9 is most closely related to the ovine DQA2 genes among sequences from various mammalian species. By contrast, the BoLA-DQA genes previously isolated are more closely related to ovine DQA1 than to the BoLA-DQA2 gene, and they represent BoLA-DQA1 genes. Thus, the presence of two BoLA A genes, which may be expressed and functional in the bovine, as well as in sheep was confirmed. A large number of amino acids unique to products of DQA2 genes of bovine and ovine origin were identified when the predicted amino acid sequences for both species were compared, and most of the DQA2-specific residues were located in the alpha 1 domain and were conserved with respect to products of DQA1 genes of ruminants. Thus, several characteristics of the bovine DQA genes were found to differ from those of human and rodent genes, despite similarities in gene structure and in nucleotide sequence.
Significant amount of 45-kDa polypeptide was found to be present in the cultured medium of chick aortic smooth muscle cells. The polypeptide as well as tropoelastin (65-kDa) reacted with monoclonal antibody for tropoelastin. Pulse-chase experiments revealed that the relative density of the 45-kDa polypeptide to tropoelastin increased with chase periods. Partially purified radioactive tropoelastin (65-kDa) was converted to a 45-kDa polypeptide fragment in the test tube. The processing from the 65- to the 45-kDa polypeptide in the test tube was inhibited by ethylenediaminetetraacetic acid but not by N-ethylmaleimide and aminophenylmethylsulfonyl fluoride. These results indicate that the 45-kDa fragment is a degradation product of tropoelastin and that processing is mediated by enzymatic cleavages with metal proteinase. Fully hydroxylated tropoelastin treated with ascorbic acid was more resistant to the enzymes than underhydroxylated tropoelastin with scorbutic condition, suggesting that the structural stability of tropoelastin is also involved in the processing rate.
Tropoelastin expression in vascular smooth muscle cells during serial cell passages from the primary to the tertiary culture was studied. The level of tropoelastin was found to be greatly reduced as the number of cell passages increased. The translational activity and level of elastin mRNA were essentially unchanged throughout the cell passages. The reduction in tropoelastin expression was not due to the repetitive trypsin treatment nor to the prolyl hydroxylation level of the newly-synthesized elastin. A comparable decline in tropoelastin expression was also found with increasing cell division in the primary cultures plated at different cell densities. A pulse-chase experiment revealed that the newly-synthesized elastin in the tertiary culture degraded more rapidly than that in the primary culture. The culture medium harvested from the tertiary culture exhibited a higher tropoelastin-degrading activity than that from the primary culture in the test-tube. The degrading activity of the tertiary culture was inhibited by the addition of 1 mM ethylenediaminetetraacetic acid or 1 mM phenylmethylsulfonyl fluoride, but not by 1 mM N-ethylmaleimide. These results suggest that the reduction in tropoelastin expression during the cell passages from the primary to the tertiary culture is due to the enhanced tropoelastin-degrading activity of the tertiary culture. The transition to tropoelastin-degrading phenotype during cell passages may explain the biological mechanisms of smooth muscle cell migration from the media to the intima observed in the pathological conditions.
gp130, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6) complex and transduces signals. To examine the role of gp130 signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 in combination with other cytokines on purified human umbilical cord blood CD34+ cells, using methylcellulose clonal assay and suspension culture in the presence or absence of serum. A combination of sIL-6R and IL-6 (sIL-6R/IL-6), but not sIL-6R or IL-6 alone, was found to dramatically stimulate expansion of hemopoietic progenitor cells as well as CD34+ cells in the presence of stem cell factor. Significant generation of multipotential hemopoietic progenitors over a period of 3 weeks in suspension culture and efficient formation of colonies, especially multilineage and blast cell colonies, in methylcellulose assay supplemented with a combination of sIL-6R/IL-6 together with stem cell factor were observed in serum-containing and serum-free culture. Addition of anti-gp130 monoclonal antibodies or anti-IL-6R monoclonal antibodies to the above cultures dose-dependently inhibited the expansion of progenitor cells in suspension culture and also completely blocked the formation of multilineage colonies in methylcellulose culture. These findings demonstrated that the significant expansion of human primitive hemopoietic progenitors could be achieved with the gp130 and c-Kit signalings initiated by the sIL-6R/IL-6 complex in the presence of stem cell factor and suggested the possible application of this method for ex vivo expansion of CD34+ cells for bone marrow transplantation.
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BACKGROUND: Since Brockhurst reported the connection between uveal effusion in nanophthalmic eyes and their scleral alterations and treated them with vortex vein decompression or sclerectomy, many observers have found abnormal accumulation of glycosaminoglycans (GAGs) in nanophthalmic sclera. These GAG abnormalities were thought to effect the collagen changes, though it was not clear which GAGs were changed. METHODS: GAGs were isolated and their contents were determined in scleral specimens from three nanophthalmic patients and five age-matched controls, using electrophoresis and the cetylpyridinium method. RESULTS: Hyaluronic acid, dermatan sulfate and chondroitin sulfate were the major GAGs in both nanophthalmic and control samples. Nanophthalmic sclera showed 2.4-fold, 10-fold and 5.5-fold increases in hyaluronic acid, dermatan sulfate and chondroitin sulfate, respectively, compared with the controls. CONCLUSION: The results suggest that increased levels of GAGs, particularly of dermatan sulfate and chondroitin sulfate may contribute to the abnormalities of collagen fibrillogenesis and be closely involved with the pathogenesis of nanophthalmos.