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Biomedical subjects

K Tamai

Publications and source records attributed to K Tamai.

At least 163 records · Page 9Linked to original sources
PubMed1994

Molecular biology of the 230-kD bullous pemphigoid antigen. Cloning of the BPAG1 gene and its tissue-specific expression.

The 230-kD bullous pemphigoid antigen is a hemidesmosomal protein of the cutaneous basement membrane zone. We have previously cloned overlapping cDNAs corresponding to the human 230-kD bullous pemphigoid antigen gene (BPAG1), located at the human chromosomal locus 6p11-12. Utilizing the cDNA clones, a genomic DNA lambda FIX II phage library was screened. Seven over-lapping genomic clones, spanning approximately 20 kb, were isolated. These clones were shown to contain the entire approximately 9-kb coding sequence of BPAG1, and it consisted of 22 separate exons which varied from 78 to 2,810 bp in size. Elucidation of 2.6 kb of 5'-flanking DNA was found to contain several putative transcriptional response elements, and development of promoter chloramphenicol acetyltransferase (CAT) reporter gene constructs allowed identification of putative cis-elements which confer keratinocyte-specific expression to the gene. In particular, a putative AP2-binding sequence (KRE2) in the position -(1,786-1778) was shown to be responsible for marked enhancement of the endogenous promoter, as well as of a heterologous thymidine kinase/CAT construct, activity in normal human keratinocytes. Normal human keratinocyte nuclear extracts contained a protein, designated as KTP1, which complexed with the KRE2 oligomer by gel mobility shift assays. UV cross-linking and Southwestern analysis suggested that KTP1 is a DNA-binding protein clearly distinct from AP2, and this protein may be responsible for the basal keratinocyte-specific expression of the BPAG1 gene.

Autoantigens↗
PubMed1994

Interleukin-10 modulates type I collagen and matrix metalloprotease gene expression in cultured human skin fibroblasts.

IL-10, originally isolated from mouse helper T cells, is a cytokine with regulatory functions on a number of interleukins. In this study we show that recombinant human IL-10 affects the expression of several genes involved in extracellular matrix synthesis and remodeling in human dermal fibroblast cultures. As judged by Northern blot analyses, type I collagen gene expression was downregulated, while collagenase and stromelysin gene expression were markedly enhanced by IL-10. No effect on tissue inhibitor of metalloproteases mRNA levels was noted. Transient transfections of skin fibroblasts with type I collagen promoter/chloramphenicol acetyl transferase reporter gene constructs showed downregulation by IL-10, suggesting inhibition at the transcriptional level. When compared with control cultures, incubation with IL-10 resulted in a decrease in immunostaining of fibroblast cultures with antibodies to human type I collagen. In contrast, immunostaining of such IL-10-treated cultures with antibodies to human collagenase resulted in an increase in immunostaining. This study suggests a role for IL-10 in the breakdown and remodeling of the extracellular matrix.

Blotting, Northern↗
PubMed1994

[Hypothermic effects on uveal blood flow and postoperative inflammation in vitrectomy].

Hypothermic effects on uveal blood flow and postoperative inflammation were evaluated in experimental vitrectomy on albino rabbits. Solutions used for intraocular perfusion were maintained at 9 degrees C and 22 degrees C. Following the vitrectomy, the rabbits' intraocular spaces were irrigated for 60 minutes. The temperature at various sites and the uveal blood flow were measured before and during the procedure. Aqueous protein concentrations were checked on postoperative days 1, 7, and 14. Another solution maintained at 37 degrees C was used as a control. There was a larger decrease in temperature at the retina than there was at the choroid and the ciliary body. Blood flow at the ciliary body decreased to 76.0% and 77.0% at the choroid after 60 minutes of irrigation at 9 degrees C. The decrease at 22 degrees C were 91.0% and 88.3%, respectively. Aqueous protein concentrations at both 9 degrees C and 22 degrees C were significantly lower than at 37 degrees C on the first postoperative day in the eyes irrigated for 60 minutes. In the eyes irrigated for 30 minutes, however, no significant differences were seen. Hypothermia during a prolonged vitrectomy operation seems to decrease inflammation in early postoperative stages.

Animals↗
PubMed1994

Peroneal arteriovenous fistula and pseudoaneurysm formation after blunt trauma. A case report.

A 56-year-old man sustained blunt trauma to the right calf which, 15 months later, manifested as a painful soft tissue mass with aggressive destruction of the fibula. Contrast-enhanced computed tomography and popliteal arteriography disclosed a peroneal arteriovenous fistula and pseudoaneurysm. Five years after pseudoaneurysm repair and fistula ligation, the patient was free of symptoms and had no further bone destruction.

Aneurysm↗
PubMed1994

Identification and cDNA cloning of single-stranded DNA binding proteins that interact with the region upstream of the human c-myc gene.

We have previously reported that a c-myc protein complex binds to the region upstream of the c-myc gene, where exist an origin of cellular DNA replication (ori) and a transcriptional enhancer. Both functions require a 21 bp long sequence, while the c-myc protein complex recognizes a 7 bp consensus therein. It was recently reported that single-stranded DNA binding proteins bound specifically to sequences that play roles in DNA replication or transcription. We examined for proteins binding to the single-stranded DNAs of the 21 bp element (myc(H-P)21). In a band shift assay with HL60 cells nuclear extract, probes of either the plus strand or the minus strand gave rise to specific signals. Mutation introduced within a short consensus (A/TCTA/TA/TT) present in both strands completely abolished binding in either case. Southwestern blotting analysis showed that proteins of molecular weight 105, 80, 50, 45, 40, 39.5 and 14 kDa bound sequence-specifically to either strand and 22 kDa to minus strand to the cognate A/TCTA/TA/TT consensus. These single-stranded DNA binding proteins were named MSSP, c-myc gene single strand binding proteins. We attempted to isolate the cDNAs encoding these proteins by screening a human cDNA library with the plus single-stranded oligonucleotide as a probe. Among several positive clones, we have characterized one, termed MSSP-1. MSSP-1 produced in E. coli as a fusion protein with GST specifically interacted with single-stranded TCTTAT (plus myc(H-P)21) and ACT-ATT (in minus myc(H-P)21), the consensus of which can be referred to as A/TCTA/TA/TT. Sequence analysis of MSSP-1 cDNA revealed that two domains thereof are homologous to the RNA binding motifs common to several ribonucleoproteins. Interestingly, the MSSP-1/GST fusion protein specifically recognized myc(H-P)21 not only in single-stranded but also in double-stranded forms. Binding properties of MSSP-1 imply its functions in DNA replication. Furthermore, when the AT stretch in the SV40 ori core was substituted by TCTTAT, MSSP-1 promoted viral DNA replication depending on the consensus sequences.

Amino Acid Sequence↗
PubMed1994

[Decreased theophylline clearance in children with acute asthma with concurrent febrile respiratory infection].

Elevated serum levels of theophylline, a first-line anti-asthma drug, are sometimes associated with febrile respiratory infection (FRI). Although it seems to be wellknown, conflicting reports still appear in journals. We studied retrospectively theophylline clearance (th-cl) in 74 episodes of acute asthma (44 with and 30 without FRI, in patients aged 12-mos.-13-yrs.) and tried to reveal the effect of fever or pathogens on th-cl. Th-cl was estimated by a single sample technique during intravenous drip infusion of aminophylline. The results were as follows: 1) Forty four episodes with FRI included 20 viral, 10 bacterial and 2 Mycoplasma pneumoniae infections (Table 2). 2) Th-cl in 44 episodes with FRI was significantly lower than in 30 without FRI (p < 0.001). 3) Decreased tendency of th-cl in episodes with viral infection was observed, but statistically it was not significant (p < 0.1). However, the differences of th-cl among 9 pair-cases with and without viral infection were quite clear (p < 0.01). 4) Th-cl in episodes with 10 bacterial and 2 Mycoplasma infections was reduced significantly (p < 0.01). 5) There was no difference in th-cl between episodes with viral, and bacterial or Mycoplasma infections (p < 0.1). As a result, it is suggested that fever, regardless of its origin, may decrease th-cl in acute asthma with FRI.

Acute Disease↗
PubMed1993

Synaptophysin immunocytochemistry in the regenerating sprouts from the nodes of Ranvier in injured rat sciatic nerve.

Following crush injury of rat sciatic nerve, strong synaptophysin immunoreactivity was demonstrated in the regenerating sprouts that emerged from the proximal nodes of Ranvier and in their growth cones that extended through the space between Schwann cell basal lamina and myelin sheath of the parent axon. These findings suggest that synaptophysin is involved in the growth regulation of regenerating sprouts.

Animals↗
PubMed1993

Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5'-end of the gene and the 3'-untranslated region of the mRNA.

Thus far, 16 distinct vertebrate collagens (types I-XVI) have been delineated. In this study, we have cloned a mouse collagenous protein, the 180-kDa bullous pemphigoid antigen (BPAG2). Isolation of over-lapping clones, together with 5' and 3' rapid amplification of cDNA ends, allowed delineation of the entire coding sequence. The 5' and 3' ends of the mRNA transcripts were confirmed by primer extension and anchored reverse transcription polymerase chain reaction analyses. The deduced polypeptide contained 1,433 amino acids, including a collagenous domain that consisted of 13 separate segments. Computer analysis of the deduced amino acid sequence demonstrated the presence of a membrane-associated segment. Examination of the 5' end of the BPAG2 gene revealed that the 295-base pair (bp) exon 1 contained two segments of (T)13AA and TT(A)11, whereas exon 2 was shown to contain the translation initiation codon. The 3' end of the mRNA transcript identified two 6-bp inverted repeat sequences that could form a stem for a 30-bp hairpin loop followed by a series of U residues. Comparison of mouse and human BPAG2 sequences demonstrated 86% homology and the unit of evolutionary period of 4.2 million years. In summary, we have cloned full-length mouse BPAG2 cDNA sequences that encode a collagenous polypeptide. We propose that this polypeptide be designated as the alpha 1-chain of type XVII collagen.

Amino Acid Sequence↗
PubMed1993

Hybrid cell extinction and re-expression of Oct-3 function correlates with differentiation potential.

The Oct-3 gene is expressed in highly undifferentiated cells and is implicated in mammalian early embryogenesis. We have generated a series of hybrid cells between pluripotent embryonal carcinoma cells (Oct-3+) and fibroblasts (Oct-3-), and have studied the regulation and function of Oct-3. Upon fusion, the hybrid cells differentiated to nestin+/Brn-2+ cells resembling neuroepithelial stem cells. Expression of Oct-3 was extinguished at the transcriptional level in all the hybrid cells examined. The Oct-3 modulating activity required for the Oct-3-mediated enhancer activation was also extinguished. When the Oct-3 transactivating function was introduced into the hybrid cells, they transformed into morphologically distinct nestin-/Brn-2- cells ('revertants'). When the 'revertant' cells subsequently lost Oct-3 expression, they differentiated back to nestin+/Brn-2+ cells. The close correlation between the phenotypic changes and the gain/loss of Oct-3 function indicates that Oct-3 can induce dedifferentiation of the neural cells.

Animals↗
PubMed1993

Identification of cysteine residues involved in disulfide formation in the inactivation of glutathione transferase P-form by hydrogen peroxide.

We previously reported that rat glutathione transferase P-form (GST-P) is inactivated by hydrogen peroxide (H2O2). This involves formation of intra- or intersubunit disulfides, at least three extra bands with molecular masses of 21.5, 18, and 37 kDa being exhibited in addition to the native subunit band of 23.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In the present study, GST-P mutants whose cysteine residues were independently substituted with alanine (C14A, C47A, C101A, and C169A) by site-directed mutagenesis were used to identify the cysteine residues responsible for the disulfide bond formation. C14A and C169A were much more inactivated than native GST-P by 1 mM H2O2, whereas C47A and, especially, C101A appeared insensitive to H2O2. On SDS-PAGE, the 21.5-kDa band was not detected in either C47A or C101A. Hydrogen peroxide treatment of mouse GST II, highly homologous to rat GST-P but possessing glycine instead of cysteine at the 101st residue, did not result in generation of the 21.5-kDa band and was also associated with less inactivation. This band was therefore considered to be due to an intrasubunit disulfide bond between Cys-47 and Cys-101. The 37-kDa band was suggested to be due to the formation of intersubunit disulfide bonds between Cys-47 residues in different subunits. Thus the Cys-47 residue together with Cys-101 may be located in an important region for GSH binding, disulfide bond formation between these residues resulting in steric hindrance.

Animals↗
PubMed1993

Changes in carpal tunnel shape during wrist joint motion. MRI evaluation of normal volunteers.

This study evaluated dynamically the morphology of the carpal tunnel during the wrist motion. The object was to determine the transverse dimension (TD), anteroposterior (AP) dimension and cross-sectional area (CA) of the carpal tunnel in the neutral, flexed and extended positions. The wrists of eight male and eight female volunteers aged 20 to 38 years were studied. With wrist extension, AP and CA decreased while the TD increased at the pisiform level, and CA increased at the hamate level. With flexion, TD and CA decreased at the pisiform level and CA decreased at the hamate level. The results showed that the median nerve may be subjected to significant pressure at the pisiform level when the wrist is extended. During wrist flexion, the cross-sectional area decreased at both the level of the pisiform and the hook of the hamate.

Adult↗
PubMed1993

Simultaneous and separated culture of keratinocytes and fibroblasts on each side of a collagen membrane.

We have developed a new cell culture system which is of benefit for the research of dermal-epidermal interaction. In this system, normal human keratinocytes are cultured on the upper surface of a permeable collagen membrane, on the undersurface of which human fibroblasts are simultaneously and separately cultured in a lifted condition. Both the keratinocytes and fibroblasts showed good proliferation and differentiation which were revealed by phase contrast microscopy as well as light and electron microscopies. A permeability test of the collagen membrane used in the present study showed that peptides of less than 30 kDa are able to penetrate through the membrane. Using this system, we estimated the total protein content and cornified envelope formation in the keratinocytes cultured with or without fibroblasts. We found that the total protein and cornified envelope formation were significantly increased when keratinocytes were cultured together with fibroblasts. When keratinocytes were cultured with fibroblasts in the condition of air-liquid interface, synthesis of the cornified envelope was further enhanced. These results indicate that fibroblasts really effect not only the proliferation but also the differentiation of keratinocytes, and the air-liquid interface enhances their effect on differentiation. This bi-phase separated cell culture system is a useful tool for the study of keratinocyte-fibroblast interaction.

Cells, Cultured↗
PubMed1993

Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.

Ammonium Sulfate↗
PubMed1993

The human 230-kD bullous pemphigoid antigen gene (BPAG1). Exon-intron organization and identification of regulatory tissue specific elements in the promoter region.

The 230-kD bullous pemphigoid antigen (BPAG1), a hemidesmosomal protein, is encoded by a gene at the human chromosomal locus 6p11-12. We have elucidated the exon-intron organization of the entire human BPAG1 gene, including approximately 2.6 kb of 5'-flanking DNA. Seven overlapping genomic clones, spanning approximately 20 kb, contained the entire approximately 9 kb coding sequence of BPAG1 and consisted of 22 separate exons, which varied from 78 to 2,810 bp in size. The 5' flanking region of DNA, upstream from the ATG initiation codon for translation, was found to contain several putative transcriptional response elements. Most interestingly, two motifs potentially conferring keratinocyte specific expression to the gene were detected. The presence of such elements was suggested by approximately 20-fold higher expression of a promoter/chloramphenicol acetyl transferase (CAT) construct in normal human epidermal keratinocytes that express the endogenous gene, as compared to several non-expressing cell types. Transient transfections with 5'-deletion clones of the promoter/reporter gene (CAT) constructs identified a region containing a putative tissue specific element, KRE2, which also conferred tissue specificity to the expression of the truncated promoter downstream from this element, however, a mutated derivative of KRE2 was not functional. Detailed knowledge of the structure and regulation of the BPAG1 gene will aid in further elucidation of diseases affecting the cutaneous basement membrane zone.

3T3 Cells↗
PubMed1993

The use of magnetic resonance imaging in diagnosing infantile neuroaxonal dystrophy.

We used MRI to investigate the brains of four children ranging from 3 to 10 years of age with infantile neuroaxonal dystrophy. T2-weighted imaging revealed characteristic findings of marked cerebellar atrophy and diffuse hyperintensity of the cerebellar cortex. At autopsy, one child had extensive astrogliosis and neuronal loss with shrinkage of the cerebellar cortex, in addition to typical widespread changes of neuroaxonal dystrophy. The characteristic hyperintensity of the cerebellar cortex on T2-weighted imaging probably is secondary to the extensive gliosis and shrinkage of the cerebellar cortex. These cerebellar findings on MRI may permit early diagnosis of infantile neuroaxonal dystrophy.

Atrophy↗
PubMed1993

[Ocular blood flow and prostaglandin E1].

Blood pressure of rabbits was decreased 20-30% by intravenous administration of prostaglandin E1. The blood flows of the ciliary body and choroid were measured using the hydrogen gas clearance method. The ciliary body blood flow was 92.6 +/- 9.3 ml/min/100 g before administration, and 91.6 +/- 18.8 after. The choroidal blood flow was 75.6 +/- 10.0 and 73.9 +/- 6.6, respectively. There were no significant differences in blood flow before and after administration of prostaglandin E1. Partial pressure of arterial blood carbon dioxide and oxygen, pupil size and ocular tension were also measured against time. Ocular tension fell slightly from 45 to 60 minutes after administration, but the other levels showed no significant differences. Since ocular blood flow displayed no significant changes in spite of the decreased systemic blood pressure, prostaglandin E1 may be useful as a hypotensive drug in ophthalmic surgery.

Alprostadil↗