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Biomedical subjects

K Tamai

Publications and source records attributed to K Tamai.

At least 19 recordsLinked to original sources
PubMed1995

Molecular cloning of a human protein that binds to the retinoblastoma protein and chromosomal mapping.

We have isolated distinct clones for cellular proteins that bind to the retinoblastoma protein by direct screening of cDNA expression libraries using purified pRB as a probe. The total nucleotide sequence of one of these clones, RBQ-3, was determined and found to encode a protein of 66 kDa localized in the nucleus. The RBQ-3 preferentially binds to underphosphorylated pRB. The region used for binding to this protein was mapped to the E1A-binding pocket B of pRB, which has sequence similarity to the general transcription factor TFIIB. We have mapped the gene to 1q32 using polymerase chain reaction analysis on a human-hamster hybrid cell panel and chromosomal fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1995

Tissue-specific expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1). Identification of a novel keratinocyte regulatory cis-element KRE3.

The 230-kDa bullous pemphigoid antigen gene (BPAG1) is expressed exclusively in basal keratinocytes of epidermis. In this study, we have identified a novel cis-element, keratinocyte responsive element 3 (KRE3), at position -216 to -197 of the human BPAG1 gene. A promoter-CAT construct containing this element had approximately 50-fold higher expression than a similar construct devoid of this sequence when tested in transient transfections of cultured human keratinocytes. However, there was no effect on the low base-line level of expression in cultured skin fibroblasts. KRE3 contains a palindromic sequence 5'-CAAATATTTG-3', and mutations in this sequence significantly reduced the promoter activity. Gel mobility shift assays with an oligomer containing KRE3 sequence demonstrated binding activity with nuclear proteins isolated from keratinocytes. One of the DNA/protein complexes was clearly specific, since competition with > 12.5-fold excess of the unlabeled oligomer resulted in disappearance of this band. No specific binding activity was noted with nuclear proteins extracted from fibroblasts. Thus, KRE3 appears to serve as the binding site for keratinocyte-specific trans-activating factor(s), and KRE3 may thus confer the tissue-specific expression to the BPAG1 gene.

Autoantigens↗
PubMed1995

Interferon-gamma-mediated inactivation of transcription of the 230-kDa bullous pemphigoid antigen gene (BPAG1) provides novel insight into keratinocyte differentiation.

Interferon-gamma (IFN-gamma) has been shown to regulate epidermal keratinocyte growth and differentiation. In this study, we examined the effects of recombinant human IFN-gamma on the expression of the gene encoding the 230-kDa bullous pemphigoid antigen (BPAG1), a marker of the mitotic basal cell phenotype in the epidermis. Northern analysis revealed a dose- and time-dependent suppression of BPAG1 expression by IFN-gamma in cultured human keratinocytes from several different donors, and incubation of the cells with IFN-gamma in the presence of cycloheximide demonstrated that this effect required ongoing protein synthesis. The inhibition of BPAG1 gene expression was also demonstrated at the protein level by indirect immunofluorescence using a monoclonal antibody recognizing the human 230-kDa bullous pemphigoid antigen. Transient transfections of cultured keratinocytes with BPAG1 promoter-chloramphenicol acetyltransferase reporter gene plasmids indicated marked suppression of the promoter activity by IFN-gamma, and deletion constructs were able to identify a defined region containing the responsive element (IFN-gamma inhibitory element). Reduced transcription of the BPAG1 gene by IFN-gamma was also demonstrated by in vitro nuclear run-on assays. These data, which indicate inactivation of transcription of a basal keratinocyte-specific gene of transcription of a basal keratinocyte-specific gene (BPAG1) by IFN-gamma, provide novel insight into the mechanisms of IFN-gamma-mediated keratinocyte gene regulation and epidermal differentiation in inflammatory diseases.

Autoantigens↗
PubMed1995

Distribution of protein kinase C (alpha, beta, gamma subtypes) in normal nerve fibers and in regenerating growth cones of the rat peripheral nervous system.

The distribution of protein kinase C (alpha, beta, gamma subtypes) was studied using immunocytochemical techniques in normal nerve fibers and in regenerating sprouts (growth cones) from the nodes of Ranvier following crush injuries to the rat peripheral nervous system. In normal nerves, for each protein kinase C subtype, immunoreactivity was present in both myelinated and unmyelinated axons. In myelinated axons, immunoreactivity for all three subtypes was patchy in the axoplasm and diffuse in the subaxolemmal peripheral zones. No immunoreactivity was found in the microtubule and neurofilament (cytoskeletal) domain. In contrast, in unmyelinated axons, immunoreactivity was distributed diffusely in the axoplasm. Schwann cells of myelinated fibers exhibited protein kinase C immunoreactivity, but those of unmyelinated fibers did not. In regenerating nerves, early sprouts and growth cones extending through the crushed site along Schwann cell basal laminae exhibited intense immunoreactivity for all three subtypes. Immunoreactivity was distributed diffusely throughout the axoplasm of the regenerating sprouts (growth cones), in which microtubules and neurofilaments were very rare. Thus, the subcellular localization of the protein kinase C immunoreactivity in growth cones of early regenerating nerves differed from that of normal parent axons. These findings suggest that protein kinase C (alpha, beta and gamma subtypes), whose subcellular distribution becomes more extensive in regenerating axons, may have important functional roles in axonal sprouting and in the regulation of growth cone activity in the peripheral nervous system.

Animals↗
PubMed1995

Characterization of myotonic dystrophy kinase (DMK) protein in human and rodent muscle and central nervous tissue.

Myotonic dystrophy (DM) is the most common form of inherited neuromuscular disease in adults and is characterized by progressive muscle wasting and myotonia. The mutation responsible for DM has been identified as the amplification of a polymorphic (CTG)n repeat in the 3' untranslated region of a gene encoding a serine/threonine kinase (DMK). We have produced a polyclonal rabbit antibody preparation against a fusion protein encoding the C-terminal amino acids 471-629 of the human DMK gene. This antibody specifically detects products of both full length and truncated human DMK genes expressed in bacteria and in insect cells. On immunoblots, we observed protein species of approximately 74 and 82 kDa in cardiac muscle, skeletal muscle, ependyma and choroid plexus. By immunofluorescence, DMK was found to localize post-synaptically at the neuromuscular junction of skeletal muscle, at intercalated discs of cardiac tissue and at the apical membrane of the ependyma and choroid plexus. We have also detected two to three species (approximately 45-50 kDa) in other regions of the brain. Synaptic localization of DMK in the cerebellum, hippocampus, midbrain and medulla was noted. These results suggest that DMK plays a specialized role in intercellular communication.

Aged↗
PubMed1995

Interferon-gamma coordinately upregulates matrix metalloprotease (MMP)-1 and MMP-3, but not tissue inhibitor of metalloproteases (TIMP), expression in cultured keratinocytes.

Matrix metalloproteases (MMP) constitute a family of proteolytic enzymes degrading extracellular matrix components. Their activity is inhibited by tissue inhibitors of metalloproteases (TIMP). Previous studies have demonstrated that various cytokines can modulate MMP and TIMP gene expression. In this study, we demonstrate that interferon-gamma coordinately upregulates MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) gene expression in cultured keratinocytes, as determined at the mRNA steady-state levels, and this effect is dependent on on-going protein synthesis. In contrast, there was no effect on TIMP-1 gene expression. Enhanced MMP-1 expression by IFN-gamma was also demonstrated at the protein level by Western analysis. Transient transfections with MMP-1 and MMP-3 promoter/reporter gene constructs revealed no response to IFN-gamma, whereas incubation of keratinocytes with this cytokine appeared to stabilize the MMP-1 mRNA, resulting in reduced turnover of the transcript. These data suggest that IFN-gamma enhances MMP gene expression at the post-transcriptional level. The altered MMP expression by IFN-gamma without concomitant effect on TIMP gene expression potentially leads to imbalance between these proteases and their inhibitors, and enhanced proteolytic activity may play a role in the remodeling of cutaneous tissue involving inflammatory processes, such as wound healing.

Adult↗
PubMed1995

Effects of local hypothermia on uveal blood flow and postoperative inflammation in vitrectomy.

Effects of local hypothermia on uveal blood flow and postoperative inflammation were evaluated in experimental vitrectomy in albino rabbits. Solutions used for intraocular perfusion were maintained at 9 degrees C, 22 degrees C or 37 degrees C. Following closed vitrectomy, the vitreous cavity was irrigated for 60 minutes. Temperatures at various sites and uveal blood flow were measured before and during the procedure. Aqueous protein concentrations were checked on postoperative days 1, 7 and 14. There was a larger decrease in temperature at the retina than at the choroid or the ciliary body. Blood flow at the ciliary body was reduced to 76.0% and that at the choroid to 77.0% of the preoperative level after 60 minutes of irrigation at 9 degrees C. The decrease was 91.0% and 88.3%, respectively, after 60 minutes of irrigation at 22 degrees C. Aqueous protein concentrations in the 9 degrees C and 22 degrees C groups were significantly lower than those in the 37 degrees C group 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. Local hypothermia during prolonged vitrectomy seems to decrease inflammation in the early postoperative stage.

Animals↗
PubMed1994

Dynamic magnetic resonance imaging for the evaluation of synovitis in patients with rheumatoid arthritis.

OBJECTIVE: To investigate whether the findings of magnetic resonance imaging (MRI) reflect rheumatoid synovitis. METHODS: Dynamic imaging enhanced with gadolinium-diethylenetriamine pentaacetic acid was performed on 10 affected knees of 9 patients with rheumatoid arthritis. Changes in signal intensity were correlated with pathologic findings in synovial biopsy specimens obtained during total knee arthroplasty. RESULTS: Enhancement was greater in regions with a higher degree of fibrin exudation, cellular infiltration, villous hypertrophy, vascular proliferation, and granulation formation. CONCLUSION: Dynamic MRI can be used for assessing local disease activity in rheumatoid synovium.

Aged↗
PubMed1994

Substance P- and calcitonin gene-related peptide-immunofluorescent nerves in the repair of experimental bone defects.

Healing of an experimental bony defect in the rat's tibia was studied with an immunofluorescent technique to clarify when and where substance P (SP) and calcitonin gene-related peptide (CGRP) would develop. The normal tibia showed a few SP- and CGRP-immunofluorescent nerve fibres. In the experimental tibia, the number of these fibres increased on the 6th day after operation, reached a peak of proliferation on the 15th day and reverted to normal after the 24th day. The changes were associated with the development and decay of callus tissue suggesting that harmful stimuli from the injured site in a bone could be mediated by sensory nerves throughout the repair period. Most of the SP- and CGRP-immunofluorescence was seen near the vessels, frequently in the same nerve fibres. The SP- and CGRP-immunofluorescent nerves seemed to take part jointly in callus formation through the enhancement of local blood flow.

Animals↗
PubMed1994

Familial positive ulnar variance with secondary radioulnar joint arthritis and extensor tendon ruptures.

A familial pattern of extensor tendon ruptures due to distal radioulnar osteoarthritis with underlying positive ulnar variance is described. Spontaneous ruptures occurred in four members of the family (one woman and three men) in middle age. Laboratory data did not indicate any sign of rheumatoid arthritis. All cases revealed significant positive ulnar variance and distal radioulnar osteoarthritis. Younger members of the family demonstrated positive ulnar variance although without osteoarthritic changes. The tendon ruptures presumably resulted from a hereditary disorder in conjunction with degenerative changes brought about by age.

Adolescent↗
PubMed1994

Enhanced elastin and fibrillin gene expression in chronically photodamaged skin.

Cutaneous aging consists of chronologic aging as well as actinic damage, referred to as photoaging. Most of the morphologic changes associated with an aged appearance result from actinic damage to the skin. The morphologic changes in sun-damaged skin are associated with accumulation of material having the staining characteristics of elastin, known as solar elastosis, in the superficial dermis. Previous studies have demonstrated the presence of elastin within areas of solar elastosis; however, little is known about the mechanisms leading to elastin accumulation in photoaged skin. In addition, fibrillin, the fibrillar component of elastic fibers, has been found in small amounts in solar elastosis. In this study we demonstrate increased elastin mRNA levels in photoaged skin, as well as increased elastin and fibrillin mRNAs in skin explant-derived fibroblasts using Northern hybridizations, compared with controls from sun-protected sites of the same individual. Increased elastin mRNA levels result from transcriptional upregulation of the gene, as demonstrated by transient transfections with a human elastin promoter/chloramphenicol acetyltransferase construct. Elevated mRNA levels were also correlated with increased elastin and fibrillin deposition in paired biopsy specimens from photodamaged and non-sun-exposed skin, as demonstrated by immunohistochemical staining. Thus, approaches to counteract transcriptional activation of elastin gene expression may be useful in preventing the changes associated with cutaneous photoaging.

Aged↗
PubMed1994

Mouse 230-kDa bullous pemphigoid antigen gene: structural and functional characterization of the 5'-flanking region and interspecies conservation of the deduced amino-terminal peptide sequence of the protein.

The 230-kDa bullous pemphigoid antigen is a hemidesmosomal protein of the cutaneous basement membrane zone. The primary sequences deduced from full-length human cDNAs predict that this molecule consists of a central rod region and flanking globular domains. To get insight into regulation of the 230-kDa bullous pemphigoid antigen gene (BPAG1), and to evaluate evolutionary conservation of the amino-terminus of the protein, we screened a mouse genomic DNA library with a 0.3-kb cDNA corresponding to the 5' end of the human 230-kDa bullous pemphigoid antigen cDNA. A positive clone was isolated, and Southern analysis of the clone with the 0.3-kb cDNA allowed isolation of a 3.0-kb Hind III fragment containing the 5' end of the coding sequence. Alignment of the sequences of this subclone and human BPAG1 sequences revealed that this fragment contained 2466 bp of 5'-flanking DNA, upstream from the ATG translation initiation site, and 258 bp of translatable sequences that encode a putative polypeptide of 86 amino acids at the amino-terminus of the protein. This deduced polypeptide showed 91% homology with the corresponding human sequence. The TATAAA and CCAAT consensus sequences, as well as several putative cis-regulatory elements, were identified in the 5'-flanking region of the mouse DNA. To test the functional promoter activity of the 5'-flanking DNA, three mouse BPAG1 promoter/CAT reporter gene constructs, with the promoter segments spanning from -1133, -525, and -213 to -1, were developed. Transient transfections of mouse transformed keratinocytes (Pam 212 cells) with these constructs revealed clearly detectable CAT activities, indicating that the 5'-flanking region contains a functional promoter. Furthermore, these experiments suggested that the upstream sequences contain upregulatory elements, as well as elements that confer, at least in part, tissue specificity to the expression of the mouse 230-kDa BPA gene.

Amino Acid Sequence↗
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↗
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↗