Biomedical subjects

R Kobayashi

Publications and source records attributed to R Kobayashi.

At least 181 records · Page 10Linked to original sources

PubMed1997

The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

Amino Acid Sequence↗

PubMed1997

Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins.

Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.

Amino Acid Sequence↗

PubMed1997

Neoagarobiose as a novel moisturizer with whitening effect.

Neoagarobiose, a disaccharide, showed a higher hygroscopic ability than glycerol or hyaluronic acid, typical moisturizing reagents. Beside, neoagarobiose whitened B16 murine melanoma cells, and showed low cytotoxicity. Therefore neoagarobiose was a rare reagent showing both moisturizing and whitening effects.

Animals↗

PubMed1997

Non-serratus-sparing antero-axillary thoracotomy with disconnection of anterior rib cartilage. Improvement in postoperative pulmonary function and pain in comparison to posterolateral thoracotomy.

To examine the usefulness of non-serratus-sparing antero-axillary thoracotomy (AAT) with disconnection of anterior rib cartilage for curative resection of lung cancer, we used retrospective analysis to compare mortality, morbidity, hospital stay, time for thoracic opening, postoperative pulmonary function, and chest pain between AAT and posterolateral thoracotomy (PLT). Subjects were 50 lung cancer patients who underwent lobectomy via AAT (n = 25) or PLT (n = 25), who were matched by sex and age. Chest pain was evaluated using a visual analog scale, a McGill pain questionnaire, and analgesic requirements up to 6 months after surgery. AAT offered adequate exposure for lobectomy and mediastinal lymph node dissection. No difference was observed between the AAT and PLT groups in postoperative mortality, morbidity, or hospital stay. Times for thoracic opening were significantly shorter in AAT than in PLT (p < 0.001). FEV1 and vital capacity 1 week after surgery were significantly preserved in patients with AAT compared with patients with PLT (p < 0.05). Chest pain was significantly reduced in AAT patients compared with PLT patients on 1 day and from 14 days to 6 months after surgery (p < 0.01 to p < 0.001). We conclude that AAT is a reasonable thoracotomy alternative to standard PLT for curative lung cancer resection, because of its adequate exposure, shortened opening time, diminished impairment of postoperative pulmonary function, and reduced chronic postoperative pain.

Cartilage↗

PubMed1997

Opening of infectious giant bulla with use of video-assisted thoracoscopic surgery.

In 5 cases, an infectious giant bulla was opened with the use of video-assisted thoracoscopic surgery (VATS). Because all bullae adhered to the thoracic wall and were noncommunicating with the airway, they were opened without complete resection, leaving their inside walls at the lung and lateral walls on the thoracic wall. The expansion of remnant lung was excellent, and postoperative air leakage did not occur in any case. The postoperative vital capacity and FEV1 improved significantly over the preoperative condition (p<0.01). Because the bronchial communication of bulla is frequently obliterated after infection within the bulla, opening of a bulla is curative and simpler, more effective, and less invasive than complete resection.

Adult↗

PubMed1997

Descending necrotizing mediastinitis secondary to pharyngitis. A case report.

The patient was a 38-year-old female with descending necrotizing mediastinitis (DNM) originating from pharyngitis. The pus from infection by Bacteroides and aerobic streptococci had collected in the anterior and posterior mediastinum and bilateral thoracic cavity. Systemic antibiotic treatment with clindamycin and gentamycin was administered promptly after admission under a diagnosis of DNM, without waiting for results of culture and sensitivity studies. Four puncture drainages were performed by echoguide to treat bilateral empyema and left upper mediastinal pus collections. Additional surgical drainage via the transcervical approach was performed to drain right upper mediastinal pus. the patient was discharged on hospital day 50, and has since been in good health. Proper drainage at all pus collection sites by echo at both aerobic and anaerobic organisms are essential in the treatment of DNM.

Adult↗

PubMed1997

Long survival after extrapleural pneumonectomy for pleural malignant mesothelioma with metastasis to infradiaphragmatic lymph node.

A woman who at the age of 47 years underwent extrapleural pneumonectomy for malignant pleural mesothelioma with metastasis to an infradiaphragmatic lymph node is alive and well 63 months later. Because such metastasis is readily caused by lymphatic tumour-cell permeation from the diaphragm and parietal pleura, the infradiaphragmatic nodes should be dissected at extrapleural pneumonectomy for malignant pleural mesothelioma.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1997

[A case of mediastinal ganglioneuroma in an elderly patient].

A 64-year-old woman was found to have a posterior mediastinal mass. X-ray findings revealed a neurogenic tumor, i.e., a schwannoma or neurofibroma. Postoperative pathological examination of the resected tumor showed it to be a ganglioneuroma an extremely rare occurrence, given the fact that the mediastinal ganglioneuroma tends to be diagnosed in adolescents and young adults.

Female↗

PubMed1997

[Long-term survival after multimodal therapy for lung cancer with pleural dissemination].

A 56-year-old man with peripheral T1N0M0 lung cancer underwent thoracotomy. Pleural dissemination was recognized intraoperatively. We performed wedge resection of the lung including the primary tumor, and applied hypotonic cisplatin treatment. We followed this up postoperatively with chemotherapy combined with cisplatin and irinotecan. One year later, a recurrent tumor occurred at the resected end of the lung, so we conducted stereotactic radiosurgery on the recurrent lung lesion, resulting in complete eradication. One year later, no relapse of cancer was observed. We conclude that implementing hypotonic cisplatin treatment in pleural dissemination of lung cancer followed by stereotactic radiosurgery on any recurrent tumor in the peripheral lung was effective for the present case.

Adenocarcinoma↗

PubMed1997

A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs.

We have purified three new human U4/U6-snRNP proteins from HeLa cells. The three proteins formed a tightly bound complex and behaved as a single species throughout the purification. All three proteins have been identified by peptide sequencing, and full-length cDNA sequences have been obtained for all of them. Two of the proteins are homologues of the Saccharomyces cerevisiae splicing factors Prp3 and Prp4, and the third protein is a cyclophilin. Both the human and S. cerevisiae Prp4 proteins have seven repeats of the WD motif and likely fold into structures very similar to those of the beta subunits of G proteins. The human Prp3 protein is highly basic and is closely related to S. cerevisiae Prp3 only in its carboxyl-terminal half. The human homologues of Prp3 and Prp4 are part of a stable complex in the absence of RNA. The third protein in the complex is a new cyclophilin. Cyclophilins have been proposed to act as chaperones in a variety of cellular processes, and we discuss some possible roles of this U4/U6 snRNP-associated cyclophilin.

Amino Acid Sequence↗

PubMed1997

[Antithymocyte globulin as conditioning regimen for bone marrow transplantation].

Bone marrow transplantation was performed with a conditioning regimen including antithymocyte globulin (ATG) for 8 patients with HLA-compatible unrelated donors or HLA mismatched donor. Administration of ATG was halted due to side effects in only 1 case, but the other cases were had no adverse reaction. During administration of ATG, platelet counts did not decrease rapidly, but platelet infusion was not effective in some cases. As compared between patients with conventional allogeneic BMT, autologous BMT or peripheral blood stem cell transplantation and those with ATG administration, no obvious difference was seen between the two groups in lymphocyte counts, CD3, CD4, CD8 and CD20 positive cells. No patient with ATG saffered graft failure or acute GVHD. However, cytomegalovirus infection was observed more frequently than in patients without ATG. In hematological malignancy, relapse was more frequent than in patients without ATG.

Adolescent↗

PubMed1996

Characterization of a high mobility group 1/2 homolog in yeast.

A 35-kDa polypeptide belonging to the high mobility group family of proteins was purified from the yeast Saccharomyces cerevisiae on the basis of its association with a DNA helicase activity. Amino acid sequence alignment suggests that this protein, Hmo1p, is related to the HMG1/2 class of chromatin-associated proteins. Consistent with this prediction, the Hmo1 protein immunolocalizes to the nucleus, binds single-stranded DNA, and unwinds DNA in the presence of eukaryotic DNA topoisomerase I. While the purified protein has no DNA helicase activity on its own, immunoprecipitation experiments confirm that Hmo1p associates with a 5' to 3' DNA helicase activity in nuclear extracts. The in vivo role of the protein was investigated by constructing an hmo1 deletion mutant. This strain has a severe growth defect, reduced plasmid stability, and chromatin that is hypersensitive to micrococcal nuclease digestion. Taken together, the data indicate that HMO1 is likely to be the homolog of HMG1/2 in higher cells and that it plays an important role in genome maintenance.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of SNAP50, a subunit of the snRNA-activating protein complex SNAPc.

The human RNA polymerase II and III snRNA promoters share a common basal element, the proximal sequence element (PSE), which is recognized by a complex we refer to as the snRNA-activating protein complex (SNAPc). Biochemical purifications suggest that SNAPc is composed of at least four polypeptides of 43, 45, 50 and 190 kDa, as well as variable amounts of the TATA box binding protein, TBP. cDNAs encoding the 43 and 45 kDa subunits, SNAP43 and SNAP45, have been isolated, but there is no evidence that either of these subunits contacts DNA. Here we report the isolation of cDNAs encoding the 50 kDa subunit of SNAPc, SNAP50. The open reading frame predicts a 411 amino acid protein, which contains two potential zinc finger motifs. Depletions with anti-SNAP50 antibodies inhibit RNA polymerase II and III snRNA gene transcription in vitro. SNAP50 interacts with SNAP43 in co-immunoprecipitation experiments, but not with SNAP45 or TBP. UV cross-linking experiments suggest that SNAP50 contacts DNA in the SNAP complex. These results are consistent with the same core SNAP complex recognizing the PSEs of RNA polymerase II and III snRNA promoters, and provide an initial view of the architecture of the SNAP complex.

Amino Acid Sequence↗

PubMed1996

HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription.

Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an approximately 350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an approximately 600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3. We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ. These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

Amino Acid Sequence↗

PubMed1996

Presence of Ca(2+)-sensitive and -insensitive SM22 alpha isoproteins in bovine aorta.

Two proteins (25- and 22-kDa) that cross-react with anti-gizzard SM22 alpha antibody were isolated from bovine aorta. The former, identical to a SM22 alpha homolog reported previously (Kobayashi, R., Kubota, T., and Hidaka, H. (1994) Biochem. Biophys. Res. Commun. 198, 1275-1280), associates with the membrane fraction in the presence of Ca2+ and dissociates in the presence of EGTA, while the latter is insensitive to Ca2+. Peptide mapping and partial sequence analysis revealed that the 22-kDa protein is an isoform of the 25-kDa protein, without 22 amino acid residues from the C-terminus. Immunological analysis of the tissue distribution of these proteins showed that the 25-kDa protein exists in lung and spleen besides aorta, while the 22-kDa protein occurs specifically in the aorta and spleen.

Amino Acid Sequence↗

PubMed1996

Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4.

Chromatin assembly factor 1 (CAF-1) assembles nucleosomes in a replication-dependent manner. The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins. There are at least two members of this subfamily in both human (p46 and p48) and yeast cells (Hat2p, a subunit of the B-type H4 acetyltransferase, and Msi1p). Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60. p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and H3 and H4, and promotes DNA replication-dependent chromatin assembly. CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps with, but is distinct from, that reported for newly synthesized H4 isolated from nascent chromatin. Our data suggest that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism.

Acetylation↗

PubMed1996

ATP-facilitated chromatin assembly with a nucleoplasmin-like protein from Drosophila melanogaster.

To gain a better understanding of the factors that can mediate chromatin assembly, we have purified and cloned a core histone-binding protein from Drosophila melanogaster embryos. This protein resembles Xenopus laevis nucleoplasmin, and it has therefore been termed dNLP, for Drosophila nucleoplasmin-like protein. dNLP is a nuclear protein that is present throughout development. Both purified native and recombinant dNLP bind to core histones and can function in the assembly of approximately regularly spaced nucleosomal arrays in a reaction that additionally requires DNA, purified core histones, ATP, and a partially purified fraction (containing at least one other assembly activity). We also analyzed the properties of an N-terminally truncated version of dNLP, termed dNLP-S, and found that the deletion of the N-terminal 31 residues of dNLP results in a loss of the specificity of the interaction of dNLP with core histones. We then compared the abilities of dNLP and Drosophila nucleosome assembly protein-1 (dNAP-1) to promote the decondensation of Xenopus sperm chromatin, a process that can be mediated by nucleoplasmin. We observed that dNAP-1, but not dNLP, was able to promote the decondensation of sperm chromatin. These and other data collectively suggest that dNLP may participate in parallel with other histone-binding proteins such as dNAP-1 in the assembly of chromatin.

Adenosine Triphosphate↗

PubMed1996

Physical interaction of mammalian CDC37 with CDK4.

CDC37 was originally identified as a Start gene in budding yeast and has been shown to be required for association of CDC28 with cyclins. The exact functional mechanism by which CDC37 promotes this association, however, remains unknown. CDK4 is a cyclin D-dependent kinase that controls progression through G1 of the mammalian cell cycle. We have detected a specific association of CDK4 with the molecular chaperon HSP90 and a 44-kDa protein that we identify as mammalian CDC37. A physical interaction between CDC37 and CDK4 suggests that CDC37 may regulate the mammalian cell cycle through a direct effect on CDK4. Association of CDK4 with both CDC37 and HSP90 may also imply a mechanistic link between the functions of CDC37 and HSP90.

Amino Acid Sequence↗