The HST-1 (fibroblast growth factor 4, FGF4) protein is a potent mitogen for a variety of cell types of mesodermal and neuroectodermal origin, including fibroblasts, endothelial cells, and melanocytes in vitro. To identify the cells and tissue targets of HST-1 in vivo, adenovirus-mediated HST-1 gene transfer was performed. In nude mice, intraperitoneal injection of 3 x 10(9) plaque-forming units of adenoviruses carrying the HST-1 gene (Adex1HST-1L) caused an increase in the number of platelets in the peripheral blood. The number of platelets reached twice the pretreated level by 12 days after the virus injection and the increased level continued up to 20-30 days thereafter. Administration of recombinant HST-1 protein resulted in a transient increase in the platelet count. The number of megakaryocytes in the bone marrow and spleen of the animals with Adex1HST-1L was increased compared with the control animals. Other hematological changes attributed to HST-1 were not observed. Although the mechanisms involved in increased levels of platelet count by HST-1 protein remain to be elucidated, these findings also suggest that adenovirus with the HST-1 gene may be efficiently used for the treatment of thrombocytopenia in various diseases.

Adenoviridae↗

PubMed1994

Characterization of RNA binding specificity of the Drosophila sex-lethal protein by in vitro ligand selection.

The Drosophila sex-lethal (Sxl) protein, a regulator of somatic sexual differentiation, is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within Sxl and transformer (tra) pre-mRNAs. To examine the Sxl RNA binding specificity in detail, we performed in vitro selection and amplification of ligand RNAs from a random sequence pool on the basis of affinity with Sxl protein. After three cycles of selection and amplification, we cloned and sequenced 17 cDNAs corresponding to the RNAs selected in vitro. Sequencing showed that most of the RNAs selected contain polyuridine stretches surrounded by purine residues. In vitro binding analysis revealed that the sequences of the in vitro selected RNAs with relatively high affinity for Sxl show similarity to that of the Sxl- and tra-regulated acceptor regions, including the invariant AG sequence for splicing. These results suggest that Sxl recognizes and preferentially binds to a polyuridine stretch with a downstream AG sequence.

Animals↗

PubMed1994

Highly frequent homozygous deletion of the p16 gene in esophageal cancer cell lines.

To study the involvement of the p16 tumor suppressor gene in esophageal cancer development, we examined homozygous deletion of the p16 gene in 13 human esophageal cancer cell lines and 9 gastric cancer cell lines, in which some of genetic alterations have already been characterized. By Southern blot analysis, homozygous deletion was observed in 12 out of the 13 esophageal cancer cell lines (92%), in 2 out of a total of 9 gastric cancer cell lines (22%). It was also found that the p16 gene loss, cyclin D1 amplification and p53 gene mutations occurred independently in these cell lines. These findings suggest that loss or mutations of the p16 gene are involved in most esophageal cancers and that mutation of this gene plays a critical role in the development of esophageal cancer.

Blotting, Southern↗

PubMed1994

Specific sequences from the carboxyl terminus of human p53 gene product form anti-parallel tetramers in solution.

Human p53 is a tumor-suppressor gene product associated with control of the cell cycle and with growth suppression, and it is known to form homotetramers in solution. To investigate the relationship of structure to tetramerization, nine peptides corresponding to carboxyl-terminal sequences in human p53 were chemically synthesized, and their equilibrium associative properties were determined by analytical ultracentrifugation. Secondary structure, as determined by circular dichroism measurements, was correlated with oligomerization properties of each peptide. The sedimentation profiles of peptides 319-393 and 319-360 fit a two-state model of peptide monomers in equilibrium with peptide tetramers. Successive deletion of amino- and carboxyl-terminal residues from 319-360 reduced tetramer formation. Further, substitution of alanine for Leu-323, Tyr-327, and Leu-330 abolished tetramerization. Circular dichroism studies showed that peptide 319-351 had the highest alpha-helix content, while the other peptides that did not form tetramers had low helical structure. These studies define a minimal region and identify certain critical residues involved in tetramerization. Cross-linking studies between monomer units in the tetramer suggest that the helices adopt an anti-parallel arrangement. We propose that conformational shifts in the helical structure of the p53 tetramerization domain result in a repositioning of subunits relative to one another. This repositioning provides an explanation relating conformational changes at the carboxyl terminus with changes in sequence-specific DNA binding by the highly conserved central domain.

Circular Dichroism↗

PubMed1994

[Roll of endothelin-1 in mechanism of cell communication system].

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Acute Kidney Injury↗

PubMed1994

Changes in blood lactate and heart rate in thoroughbred horses during swimming and running according to their stage of training.

The purpose of this study was to investigate whether the change in the performance capacity of horses trained by running could be evaluated with a standardised swimming exercise test as well as by a standardised running exercise test. Seven two-year-old thoroughbred horses were trained by running for four months and were subjected to a standardised swimming exercise tolerance test before the training began and after two and four months of training in addition to the standardised running tolerance test after two and four months of training. The running training brought about a significant change in the correlation between the swimming speed of the horses and their blood lactate concentration, and the correlation between the blood lactate concentration and maximum heart rate and running speed also changed significantly after two months of training.

Animals↗

PubMed1994

Angiogenic activity of the recombinant hst-1 protein.

The hst-1 transforming gene encodes a protein which belongs to the FGF family of growth factors. We showed previously that a human hst-1 protein produced in silkworm cells has in vitro mitogenic activity to vascular endothelial cells. Here we report effective synthesis of an unfused human hst-1 protein in E. coli and a potent in vivo angiogenic activity of this hst-1 protein by two in vivo assays for angiogenesis, chick chorioallantoic membrane assay and rat cornea assay. The NIH3T3 transformant transfected with the hst-1 gene appeared to develop a highly-vascularized tumor on nude mice. These data showed that the hst-1 protein has an angiogenic activity in vivo as well as in vitro.

Animals↗

PubMed1994

High-resolution structure of the oligomerization domain of p53 by multidimensional NMR.

The three-dimensional structure of the oligomerization domain (residues 319 to 360) of the tumor suppressor p53 has been solved by multidimensional heteronuclear magnetic resonance (NMR) spectroscopy. The domain forms a 20-kilodalton symmetric tetramer with a topology made up from a dimer of dimers. The two primary dimers each comprise two antiparallel helices linked by an antiparallel beta sheet. One beta strand and one helix are contributed from each monomer. The interface between the two dimers forming the tetramer is mediated solely by helix-helix contacts. The overall result is a symmetric, four-helix bundle with adjacent helices oriented antiparallel to each other and with the two antiparallel beta sheets located on opposing faces of the molecule. The tetramer is stabilized not only by hydrophobic interactions within the protein core but also by a number of electrostatic interactions. The implications of the structure of the tetramer for the biological function of p53 are discussed.

Base Sequence↗

PubMed1994

Interaction between cytochrome P450 2B1 and cytochrome bs: inhibition by synthetic peptides indicates a role for P450 residues Lys-122 and Arg-125.

Binding of cytochrome b5 to rat cytochrome P450 2B1 was inhibited (by 75%) by a synthetic peptide corresponding to P450 residues 116-134. The role of Lys-122 and Arg-125 were evaluated using peptides in which one or both of these basic residues were replaced with Glu. The Lys-122 substitution nearly abolished while the Arg-125 replacement decreased (by 20%) the inhibitory potential of the peptide. Substitution of both residues resulted in a peptide with no inhibitory activity. These results thus indicate a role for a specific P450 region as well as two basic residues within this region in the cytochrome P450-cytochrome b5 interaction.

Amino Acid Sequence↗

PubMed1994

Preferential alternative splicing in cancer generates a K-sam messenger RNA with higher transforming activity.

K-sam, also designated fibroblast growth factor receptor 2/BEK, was originally cloned from a stomach cancer cell line KATO-III. The gene is amplified and overexpressed preferentially in poorly differentiated types of stomach cancers. The major K-sam transcript in KATO-III cells encodes a receptor protein with a truncated carboxyl terminus and with a high-affinity binding site for keratinocyte growth factor. This truncated type is produced by an alternative splicing mechanism, and in normal tissues, the truncated type is far less prevalent than the untruncated form. The variant K-sam complementary DNA lacks tyrosine 769, which is a putative phospholipase C gamma 1 association site, and showed a higher transforming activity to NIH3T3 cells than the untruncated form, which is identical with the keratinocyte growth factor receptor.

3T3 Cells↗

PubMed1994

Human papillomavirus not found in squamous and large cell lung carcinomas by polymerase chain reaction.

BACKGROUND: Human papillomavirus (HPV) has been identified not only in anogenital carcinomas, but also in malignancies of other organs, including bronchogenic carcinomas. Previous studies reporting detection of these viruses in lung cancers used mainly in situ hybridization. The authors applied the polymerase chain reaction (PCR) for investigating the occurrence of HPV in bronchial neoplasms. METHODS: Formalin fixed, paraffin embedded tissues of 40 squamous and 7 large cell lung cancers were examined. PCR was done with consensus primers, capable of detecting HPV 6, 11, 16, 18, 31, 33, 52b, and 58. RESULTS: None of the 47 samples contained any of the examined HPV types. CONCLUSIONS: Because the squamous and large cell cancer cases were found not to be associated with HPV infection, this study does not support the potential role of these viruses in the development of lung cancers.

Base Sequence↗

PubMed1994

Search for a light scalar top squark in e+e- reactions at Ec.m.=58 GeV.

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Journal Article↗

PubMed1994

Construction of a novel database containing aberrant splicing mutations of mammalian genes.

To explore the rules for mammalian splice-site selection using a statistical approach, we constructed an aberrant splicing database containing an extensive collection of mammalian genetic disease mutations (90 genes, 209 mutations). From this database, we confirmed that: (1) more than 90% of mutations either destroy or create the splice-site consensus sequences; (2) the number of mutations mapped at individual residues in the splice-site regions roughly correlates to their conservation degrees in the consensus sequences; (3) about half of the observed aberrant splicing is exon skipping, while intron retention is rarely observed; (4) almost all of the major cryptic sites, activated by mutations, are mapped within an about 100-nt region from the authentic splice sites. Furthermore, we found that: (5) mutations are observed more frequently in the 5' splice-site region than in the 3' splice site region; (6) splice sites that are newly created by mutations are located upstream from the authentic splice sites. Hopefully, these observations will be used as rules for constructing a more effective prediction system of exon sequences.

Consensus Sequence↗