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Biomedical subjects

S Aota

Publications and source records attributed to S Aota.

At least 55 records · Page 3Linked to original sources
PubMed1995

Changes in urinary deoxypyridinoline level and vertebral bone mass in the development of adjuvant-induced arthritis in rats.

This study was designed to determine the effect of bone resorption on the development of generalized osteopenia in adjuvant-induced arthritic rats. Thirty of a total of sixty male SD rats, 6 weeks of age, were injected with killed mycobacterium butyricum suspended in mineral oil into the right hind paw and assigned to six groups of 5 animals each. The other thirty animals served as the age-matched noninjected controls. Animals were sacrificed at 4, 7, 10, 14, 21, and 28 days post-injection after measuring the bilateral hind-paw volumes. Twenty-four-hour urinary samples were obtained before sacrifice and the levels of deoxypyridinoline (D-Pyr) and creatinine (CR) were measured. Plasma intact osteocalcin levels were measured by a sandwich enzyme immunoassay at the start, 14 and 28 days after injection. Bone mineral measurement and histomorphometrical analyses were performed on specimens of the third lumbar vertebral body. On the seventh day after injection, arthritic rats showed significant decreases in the values of bone mineral content (BMC) and density (BMD) when compared to controls. Urinary D-Pyr/Cr ratios, however, did not increase on the seventh day, showing a significant increase on the tenth day after injection. The serum osteocalcin level was significantly reduced on the fourteenth day. The trabecular bone volume (BV/TV) in the arthritic rats showed a significant decrease from the seventh day. The trabecular thickness (Tb.Th) value significantly decreased on the seventh day after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗
PubMed1995

Why is there no carbonic anhydrase activity available to fish plasma?

Carbonic anhydrase (CA) is absent from the plasma of vertebrates. In vitro, CA in fish plasma will short-circuit the effect of catecholamines, which is to increase red blood cell (RBC) pH and volume, both of which enhance the affinity of hemoglobin for O2. CA was infused into trout for a period of 6 h and injected after 48 h, during which the animal was submitted to deep hypoxia (PO2=30­35 mmHg; 4.0­4.7 kPa). O2 content, lactate content, catecholamine levels, hematocrit, hemoglobin concentration and pHi were similar to those in the saline-infused control group. In contrast, cell volume was significantly higher and pHe, total CO2 content and organic phosphate levels were significantly lower than in the control group. The concentration of CA was not high enough completely to short-circuit the increase in pHi and red blood cell volume caused by catecholamines. The lower pHe in the CA-infused animals could enhance the activity of the Na+/H+ pump, which would keep the nucleotide triphosphate levels low. pH is a balance between acid loading at the muscle and acid excretion at the gills or the kidneys; we cannot distinguish between which of these resulted in a decrease of plasma pH. In conclusion, CA in plasma did not cause the expected reduction in blood oxygen content but did have a marked effect on plasma total CO2 content.

Journal Article↗
PubMed1995

Function and receptor specificity of a minimal 20 kilodalton cell adhesive fragment of fibronectin.

Previous studies have reached conflicting conclusions about the minimal size and sequences of the fibronectin cell-adhesive domain necessary for retention of high cell adhesive activity. We have expressed a recombinant 20 kDa cell-binding fragment of human fibronectin consisting of the ninth and tenth type III modules, which includes the Arg-Gly-Asp (RGD) cell recognition site and a second cell adhesive domain that acts synergistically with the RGD site. This polypeptide retained a similar activity as a larger 110 kDa fibronectin fragment when used in soluble form in inhibition assays, but it displayed low cell adhesive activity if assayed after direct adsorption to a plastic substrate. However, adhesive function was restored if the fragment was bound to a non-inhibitory anti-fibronectin antibody pre-adsorbed to the plastic substrate. The antibody-bound fragment also promoted cell migration. Both cell spreading and migration were specifically mediated by the alpha 5 beta 1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound alpha 5-, alpha 3-, and alpha v-containing fibronectin-binding integrins. In contrast, an immobilized 11.5 kDa fragment that contained the RGD sequence but lacked the synergistic sequence was bound only poorly by alpha 5-containing fibronectin receptor integrins, even though the alpha 3- and alpha v-containing integrins bound readily. Our results indicate that the manner in which adhesion proteins are presented to cells is important and that most cell adhesive activity is retained in a minimal 20 kDa segment of fibronectin.

Base Sequence↗
PubMed1994

The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function.

Synergistic sites in the central cell-adhesive domain of fibronectin (FN) substantially enhance cell adhesion mediated by the alpha 5 beta 1 integrin receptor for fibronectin. We characterized a critical minimal sequence needed for synergistic activity using site-directed mutagenesis and homology scanning using intramolecular chimeras. The minimal cell-binding domain of FN consisting of the 9th and 10th type III FN repeat was expressed in an Escherichia coli expression system. This protein retained high biological activity when assayed using a competitive inhibition assay for FN-mediated adhesion of baby hamster kidney or HT-1080 cells. In contrast, a construct consisting of the 8th and 10th repeat displayed very low biological activity. By replacing various portions of the 8th repeat with homologous 9th repeat segments, we mapped the synergistic region to the center of the 9th repeat. When a very short peptide sequence, Pro-His-Ser-Arg-Asn (PHSRN), from the 9th repeat was substituted for the homologous pentapeptide site in the 8th repeat sequence, the recombinant protein showed markedly enhanced activity. Further mutagenesis analysis suggested that the arginine residue of this pentapeptide sequence is important for function. We also identified a weaker adjacent synergy region other than the PHSRN region. Epitope mapping of an anti-FN monoclonal antibody that inhibits FN-mediated adhesion identified the same critical regions. A synthetic peptide containing the PHSRN sequence showed neither competitive inhibitory activity in solution nor synergy with a soluble RGD-containing peptide. However, when the same synthetic peptide was positioned via a covalent bond at the corresponding site of the normally inactive 8th repeat, it mediated an enhancement of adhesive activity. These results identify a pentapeptide site that synergistically enhances the cell-adhesive activity of the FN RGD sequence.

Amino Acid Sequence↗
PubMed1991

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin.

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.

Amino Acid Sequence↗
PubMed1991

Characterization of regions of fibronectin besides the arginine-glycine-aspartic acid sequence required for adhesive function of the cell-binding domain using site-directed mutagenesis.

Previous studies of adhesion mediated by the central cell-binding domain of fibronectin suggest that additional polypeptide information besides the Arg-Gly-Asp sequence is required for full activity. We analyzed this putative second, synergistic region of fibronectin more extensively by deletion analysis and oligonucleotide-based site-directed mutagenesis. Resulting mutated fusion proteins expressed using lambda gt11 were assayed for baby hamster kidney fibroblast cell spreading activity. Deletion mutants truncating from the amino terminus showed a decrease of activity in two apparently discrete steps. Complementary studies using a series of overlapped internal deletions designed to retain the repetitive fibronectin structure also indicated that two distinct peptide regions besides the RGD sequence were necessary for full activity. Removal of the carboxyl-terminal region resulted in the greatest loss of activity (greater than or equal to 20- versus 3-5-fold). Very similar results were obtained with HT-1080 cells dependent on the alpha 5 beta 1 integrin receptor for adhesion to fibronectin. An anti-fibronectin monoclonal antibody that inhibits cell adhesion was found to bind to the carboxyl-terminal functional region, and a point mutation caused specific loss of its epitope. These studies reveal unexpected complexity in the organization of these functional regions, which contrasts with adhesion models based only on simple, short peptide recognition sequences.

Amino Acid Sequence↗
PubMed1991

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.

Amino Acid Sequence↗
PubMed1990

Giant G+C% mosaic structures of the human genome found by arrangement of GenBank human DNA sequences according to genetic positions.

To determine the overall variation in the G+C% distribution over long ranges of the human genome, DNA sequences of human genes, which were closely linked genetically or physically, were surveyed from the GenBank Data Bank. A total of 72 sequences longer than 2 kb, which were mutually linked within 500 kb, were identified. The sequences belonged to 17 linkage groups and were ordered in each group according to their genetic positions. Analyses of the G+C% distribution along the ordered sequences showed that sequences within each group almost always had similar G+C% levels, but those belonging to different groups often had different levels. Similar analyses of more distantly linked sequences (e.g., greater than 10 Mb) showed mosaic structures of G+C% distribution. These findings are consistent with predictions made from the "isochore" structures found by CsCl equilibrium centrifugation, in that the structures having homogeneous base compositions stretched over at least several hundred kilobases. A possible boundary of the giant G+C% mosaic structures was identified between X-linked G6PD and F8C.

Base Composition↗
PubMed1988

Global variation in G+C content along vertebrate genome DNA. Possible correlation with chromosome band structures.

The global, rather than local, variation in G+C content along the nuclear DNA sequences of various organisms was studied using GenBank sequence data. When long DNA sequences of the genomes of Escherichia coli and Saccharomyces cerevisiae were examined, the levels of their G+C content (G+C%) were found to be within a narrow range around that of the whole genome. The G+C% levels for sequences of vertebrate genomes, however, were found to cover a wide range, showing that their genome is a mosaic of sequences with different G+C% levels, in each of which the sequence is fairly homogeneous in its G+C% for a very long distance. Through surveying a human genetic map and GenBank DNA sequences, the global variations in G+C% along the human genome DNA were found to be correlated with chromosome band structures.

Animals↗
PubMed1987

Nucleotide sequence and molecular evolution of mouse retrovirus-like IAP elements.

We determined the nucleotide (nt) sequences of cDNA and genomic clones for murine intracisternal type A particle (IAP) elements, which are retrovirus-like repetitive sequences in rodent genomes. The nucleotide sequence of the cDNA resembled that of retrovirus RNA genomes in its lack of the U5 sequence within the 3' long terminal repeat. By sequence comparison of our clones with reported rodent IAP elements, we located the probable gag, pol and env gene regions. The sequences for the pol, env and the 3' two-thirds of the gag region were conserved among the IAP elements. In the regions, synonymous substitutions occurred more frequently than non-synonymous ones, which suggested that the regions in question were functionally constrained until fairly recently. The rate of nucleotide substitutions in the regions was estimated to be 6-10 X 10(-9) nt per site per year, and significantly higher than that of the cellular genes. These rates may exemplify a characteristic of the nucleotide substitutions for an endogenous retrovirus. The sequence homology between the IAP element and IgE-binding factor gene is discussed.

Animals↗
PubMed1986

A sequence homology between the pX genes of HTLV-I/II and the murine IL-3 gene.

Searching the protein sequence database for amino acid sequences homologous to the x-lor sequence in the pX region of human T-cell leukemia virus types I and II (HTLV-I/II), we found that there is a region of 38 amino acids where the murine interleukin 3 (IL-3) sequence has a 40% homology with the x-lor sequence. A statistical analysis shows that this homology is highly significant with a probability of 1.57 X 10(-10). The biological implication of this homology is discussed.

Amino Acid Sequence↗
PubMed1986

Diversity in G + C content at the third position of codons in vertebrate genes and its cause.

Correlation was positive between the G + C content at the codon third position in genes of vertebrates and the G + C content of the genome portion surrounding each gene. Exons of genes with a high G + C% at the codon 3rd position are surrounded by G + C-rich introns and G + C-rich flanking sequences, and those with a low G + C% at the position by A + T-rich introns and flanking sequences. Analysis of G + C content distribution along DNA sequences using a DNA Sequence Data Bank supported the view that the vertebrate genome is a mosaic of regions with clear differences in their G + C content. The biological significance of the variation in G + C content throughout the vertebrate genome is discussed in connection with chromosomal banding.

Animals↗