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Biomedical subjects

M Obara

Publications and source records attributed to M Obara.

At least 73 records · Page 4Linked to original sources
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

The complete primary structure of type XII collagen shows a chimeric molecule with reiterated fibronectin type III motifs, von Willebrand factor A motifs, a domain homologous to a noncollagenous region of type IX collagen, and short collagenous domains with an Arg-Gly-Asp site.

Extracellular matrix molecules are generally categorized as collagens, elastin, proteoglycans, or other noncollagenous structural/cell interaction proteins. Many of these extracellular proteins contain distinctive repetitive modules, which can sometimes be found in other proteins. We describe the complete primary structure of an alpha 1 chain of type XII collagen from chick embryonic fibroblasts. This large, structurally chimeric molecule identified by cDNA analysis combines previously unrelated molecular domains into a single large protein 3,124 residues long (approximately 340 kD). The deduced chicken type XII collagen sequence starts at the amino terminus with one unit of the type III motif of fibronectin, which is followed by one unit homologous to the von Willebrand factor A domain, then one more fibronectin type III module, a second A domain from von Willebrand factor, 6 units of type III motif and a third A domain, 10 consecutive units of type III motif and a fourth A domain, a domain homologous to the NC4 domain peptide of type IX collagen, and finally two short collagenous regions previously described as part of the partially sequenced collagen type XII molecule; an Arg-Gly-Asp potential cell adhesive recognition sequence is present in a hydrophilic region at the terminus of one collagenous domain. Antibodies raised to type XII collagen synthesized in a bacterial expression system recognized not only previously reported bands (220 kD et cetera) in tendons, but also bands with apparently different molecular sizes in fibroblasts and 4-d embryos. The antibodies stained a wide variety of extracellular matrices in embryos in patterns distinct from those of fibronectin or interstitial collagens. They prominently stained extracellular matrix associated with certain neuronal tissues, such as axons from dorsal root ganglia and neural tube. These studies identify a novel chimeric type of molecule that contains both adhesion molecule and collagen motifs in one protein. Its structure blurs current classification schemes for extracellular proteins and underscores the potentially large diversity possible in these molecules.

Amino Acid Sequence↗
PubMed1991

Mechanisms of resistance to beta-lactam antibiotics in Acinetobacter calcoaceticus.

Examination of 12 strains of Acinetobacter calcoaceticus revealed that the strains expressed different constitutive levels of beta-lactamase. Mutants resistant to cefoxitin, cefoperazone or ceftazidime were selected from a strain producing a low level of beta-lactamase. All the mutants showed no change in expression of beta-lactamase, but produced penicillin-binding proteins with altered expression and/or affinity for beta-lactams. In addition, the outer membrane of the mutants showed decreased permeability (40-80% that of the parent strain) towards small hydrophilic solutes, together with diminished production of a 46.5 kDa porin protein. It was concluded that the enhanced resistance to beta-lactams in the A. calcoaceticus mutants was the result of interplay between the altered penicillin binding proteins and the reduced outer membrane permeability.

Acinetobacter calcoaceticus↗
PubMed1990

Hydrocortisone-induced enhancement of expression and changes in methylation of pepsinogen genes in stomach mucosa of the developing rat.

Administration of hydrocortisone to infant rats caused a precocious increase in levels of mucosal pepsinogen and its mRNA together with morphological maturation of pepsinogen-producing cells. The increase in levels of pepsinogen mRNA was induced rapidly and was associated with increase in levels of its precursors, suggesting transcriptional regulation of pepsinogen genes by hydrocortisone. Methylation analysis with the methylation-sensitive restriction enzymes, HpaII and HhaI, revealed that hydrocortisone also induced sequential demethylation changes of CCGG and GCGC sites in and around pepsinogen genes. Most of these changes occurred after increases in transcription of the genes and did not appear to play a causal role in gene activation. Superficially, the observed demethylations corresponded to the sequential processes of morphological maturation of pepsinogen-producing cells. Thus, these changes in methylation are probably linked to hydrocortisone-induced differentiation of pepsinogen-producing cells and may reflect the mechanism in vivo for the maturation of pepsinogen genes.

Animals↗
PubMed1990

[Pulsatile LH secretion and hypothalamic-pituitary function in amenorrheic women].

In order to evaluate hypothalamic-pituitary function and pulsatile LH secretion in 42 amenorrheic women, blood was collected every 20 min. for 6 hrs. The mean LH pulse frequency (LH-pf) of women with a normal cycle, PCO and hyperprolactinemia was 3.4 +/- 0.5, 5.2 +/- 0.8, and 1.6 +/- 1.0/6 hrs, respectively. Hypothalamic amenorrhea (HA) with no progesterone withdrawal bleeding showed a lower frequency of LH secretion. As LH-pf in HA with progesterone withdrawal bleeding (HAI) ranged from 0 to 5/6 hrs, the HAI group was divided into 2 groups according to whether their LH-pf was less than 3.4/6 hrs (HAIa) or more (HAIb). Pituitary response to 100 micrograms of LH-RH in HAIb was much lower than in HAIa and PCO. On the other hand, in HA with LH-pf less than 3.4, LH-pf showed a significant correlation with basal LH and FSH levels and the LH increase to 100 micrograms of LH-RH, while in HA with LH-pf greater than or equal to 3.4, LH-pf had no significant correlation with them. In PCO, a high LH pulse frequency and amplitude and LH response to 100 micrograms of LH-RH were shown, but the LH increase to 100 micrograms of LH-RH was significantly reduced with the increase in LH-pf. Pituitary response to 100 micrograms of LH-RH in women with hyperprolactinemia was maintained despite low LH-pf. These results suggest that the analysis of pulsatile LH secretion is very helpful in evaluating hypothalamic-pituitary function in amenorrheic women.

Amenorrhea↗
PubMed1990

[Clinical effects of pulsatile LH-RH administration on ovulation induction for women with various type of amenorrhea].

Using pulsatile LH-RH administration, ovulation induction was performed in women with pituitary dwarfism (PD, n = 1), isolated gonadotropin deficiency (IGD, n = 5), secondary hypothalamic amenorrhea (gestagen negative (AM2, n = 10), positive (AM1, n = 5)), polycystic ovarian disease (PCO, n = 6) and anovulatory cycle (ANOV, n = 1). Five to 20 micrograms of LH-RH was administered subcutaneously with a pulse frequency of 90 min to 2 h, in 76 treatment cycles. The ovulation rate of IGD, AM2, AM1 and PCO was 54.5, 83.3, 12.5 and 50.0%, respectively, all being significantly different from each other. In some cases, ovulation induction was repeated for several cycles without any interruption, and chronic effects of this therapy on the subsequent cycle were examined. In IGD and AM2, subsequent cycles were well stimulated, while those of PCO became refractory. These results indicate that pulsatile LH-RH administration should be the first choice of ovulation induction in IGD and AM2, and less effective in AM1 and PD. When this treatment is applied for PCO, the luteal phase should be supported by alternative methods to avoid pituitary desensitization.

Amenorrhea↗
PubMed1989

Changes in the steady-state fluorescence anisotropy of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle.

Cys674 of the sarcoplasmic reticulum Ca2+-ATPase was selectively labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and the steady-state fluorescence anisotropy of this label and its total fluorescence intensity were followed throughout the catalytic cycle. At 25 degrees C, the anisotropy and the total fluorescence intensity increased by 2.1 and 9.4%, respectively, upon Ca2+ binding to the high affinity sites. Upon subsequent ATP binding to the catalytic site, the anisotropy and the total fluorescence intensity decreased by 6.8 and 23.9%, respectively. These drops likely occurred in the enzyme.ATP complex. The extents of changes upon additions of Ca2+ and ATP in the anisotropy, but not in the total fluorescence intensity, were greatly reduced by lowering the temperature. Slight drops in the anisotropy and the total fluorescence intensity occurred upon conversion of phosphoenzyme (EP) from the ADP-sensitive form to the ADP-insensitive form. The anisotropy and the total fluorescence intensity returned to the initial level when EP was hydrolyzed. Mg2+-dependent Pi-induced drops in the anisotropy and the total fluorescence intensity occurred coincidently with EP formation from Pi. These demonstrate that the ATP-induced drops in the anisotropy and the total fluorescence intensity are predominant throughout the catalytic cycle. Most probably, the changes in the anisotropy are due to changes in the rotational diffusion of the label. These findings indicate that ATP binding to the catalytic site induces a relaxed conformation in the microenvironment of the label bound to Cys674.

Adenosine Triphosphate↗
PubMed1988

Biosynthesis of a novel transformation-sensitive heat-shock protein that binds to collagen. Regulation by mRNA levels and in vitro synthesis of a functional precursor.

The synthesis of a major collagen-binding glycoprotein of molecular weight 47,000 was previously shown to be altered by malignant transformation as well as by heat shock in chick embryo fibroblasts (Nagata, K., and Yamada, K.M. (1986) J. Biol. Chem. 261, 7531-7536 and Nagata, K., Saga, S., and Yamada, K.M. (1986) J. Cell Biol. 103, 223-229). In this paper, we examined the synthesis of this heat shock protein (hsp47) in terms of possible functional precursors and its regulation after heat shock and transformation by Rous sarcoma virus. Actinomycin D inhibited the induction of hsp47 after heat shock. Messenger RNAs purified from chick embryo fibroblasts (CEF), heat-treated CEF, and transformed CEF were analyzed in an in vitro translation system. In vitro translated products readily bound to gelatin-Sepharose, and levels were increased after heat shock and decreased after transformation. The increase in mRNA after heat shock was shown more directly by Northern assay using a synthetic oligonucleotide probe. We identified two putative precursors of hsp47 using an in vitro translation/processing system and tunicamycin: one is a 42-kDa primary translation product and the second is a 41-kDa polypeptide lacking signal peptide and carbohydrate moieties. Both of these precursors are biologically active as determined by gelatin-binding activity, in contrast to the lack of binding activity of precursors in several other membrane-associated receptor systems.

Animals↗
PubMed1988

Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function.

Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis. Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E. coli by greater than or equal to 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a greater than or equal to 96% loss of activity, indicating cooperativity between sites. The two classes of mutant protein displayed synergism of activity in a trans complementation assay. Effective actin microfilament bundle organization was also dependent on the combined function of both sites. Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.

Actins↗
PubMed1988

Conformational changes in the vicinity of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle.

In the previous experiment (Suzuki, H., Obara, M., Kuwayama, H., and Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456), the Ca2+-ATPase of sarcoplasmic reticulum vesicles was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The main labeled site was Cys674. A large monophasic fluorescence drop occurred upon ATP binding to the catalytic site of the Ca2+-activated enzyme in the presence of K+. The present results show that this fluorescence drop is biphasic in the absence of K+. The first and rapid phase of this drop accounts for most of the fluorescence drop. This phase reflects a conformational change in the enzyme.ATP complex. The second and slow phase, being much smaller than the first phase, coincides with phosphoenzyme (EP) isomerization from the ADP-sensitive form to the ADP-insensitive form. This phase disappears when accumulation of ADP-insensitive EP is inhibited by K+ or when EP isomerization is prevented by the N-ethylmaleimide treatment. These results show that this phase reflects a conformational change upon EP isomerization. When free Ca2+ is chelated after EP formation from ATP, the fluorescence intensity is restored to the initial level without Ca2+. This restoration coincides with EP decomposition. This suggests that the fluorescence restoration reflects a conformational change upon hydrolysis of ADP-insensitive EP. This probability is supported by the concurrent occurrence of the Pi-induced fluorescence drop and EP formation from Pi. The results demonstrate that the fluorescence drop upon ATP binding is predominant in the fluorescence change throughout the catalytic cycle.

Adenosine Triphosphate↗
PubMed1988

Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules.

Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell-binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively-spliced type III homology-connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.

Animals↗
PubMed1987

Genetic analysis of the cell binding domain region of the chicken fibronectin gene.

We have determined the nucleotide sequence of the cell binding domain region of the chicken fibronectin gene and analyzed it evolutionaly. We present here the complete nucleotide sequence of 4.3 kb HindIII/EcoRI segment from the clone lambda FC23 of the chicken fibronectin gene. There were five exons in this segment. When we lined up the amino acid of exons 28, 29 and 31, three alignments, known as the Type III repeat, appeared. Tetrapeptide, -RGDS-, called the cell binding domain, existed in the second repeat, coding exon 30. It was presumed that the Type III repeats were composed of two exons in the chicken gene, the same as in the rat and humans. We found repeatedly appearing amino-acid sequences such as -TIT- (three arrays in these Type III repeats) but also found one of the amino acids substituted in the tripeptide in these Type III repeats (seven arrays). We analyzed these repeats from the point of view of evolution. We used three of the nucleotide sequences (12-18 bp) coding such -TIT- repeats as a unit length for comparing the various homologies after dividing the coding region into 56 segments. The mutual homology of the divided segments to each one of three showed 53% on average. On the other hand, the mutual nucleotide homology of the Type III repeat was 44%. This suggested that the Type III repeat may have been developed by frequent duplication of small gene units.

Amino Acid Sequence↗
PubMed1987

A conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2+-ATPase upon ATP binding to the catalytic site.

Sarcoplasmic reticulum vesicles were modified with a fluorescent thiol reagent, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. One mol of readily reactive thiols per mol of the Ca2+-ATPase was labeled without a loss of the catalytic activity. The fluorescence of the label increased by 8% upon binding of Ca2+ to the high affinity sites of the enzyme. This fluorescence enhancement probably reflects a conformational change responsible for Ca2+-induced enzyme activation. Upon addition of ATP to the Ca2+-activated enzyme, the fluorescence decreased by 15%. This fluorescence drop and formation of the phosphoenzyme intermediate were determined under the same conditions with a stopped-flow apparatus and a rapid quenching system. The amplitude of the fluorescence drop thus determined was saturated with 3 microM ATP. This shows that the fluorescence drop was caused by ATP binding to the catalytic site. In contrast, the rate of the fluorescence drop was not saturated even with 50 microM ATP. The fluorescence drop coincided with phosphoenzyme formation at 0.5 or 3 microM ATP, but it became much faster than phosphoenzyme formation when the ATP concentration was raised to 100 microM. These results indicate that the ATP-induced fluorescence drop reflects a conformational change in the enzyme.ATP complex. The fluorescence drop was accompanied by a red spectrum shift, which suggests that the label was exposed to a more hydrophilic environment. The electrophoretic analysis of the tryptic digest of the labeled enzyme (10.9 kDa) showed that almost all of the label was located on the 5.2-kDa fragment which includes the carboxyl terminus and the putative ATP-binding domain. The sequencing of the two major labeled peptides, which were isolated from the thermolytic digest of the labeled enzyme, revealed that the labeled site in either of these peptides was Cys674. It seems likely that the label bound to this Cys674 could be involved in the observed fluorescence changes.

Adenosine Triphosphate↗
PubMed1987

Analysis of N-myc amplification in relation to disease stage and histologic types in human neuroblastomas.

Both untreated and treated primary neuroblastomas from 52 patients were analyzed to determine the correlation between the amplification of N-myc oncogene and various prognostic factors. Amplification of N-myc was observed in eight of 28 untreated cases and in 12 of 24 treated cases. As a whole, 12 of 18 tumors (67%) in Stage IV had N-myc amplification, but there were fewer cases in the unadvanced disease stage, as reported previously by others. Furthermore, the authors detected N-myc amplification in three of nine tumors in Stage IV-S, although the amplification was less than 50 copies. Analysis of progression-free survival at 24 months revealed that amplification of N-myc was associated with the worst prognosis (P less than 0.001). In the untreated group, no amplification of N-myc was detected in any of two ganglioneuromas and four ganglioneuroblastomas, whereas amplification of N-myc was observed in all two round-cell and six of 20 rosette fibrillary neuroblastomas. On the other hand, the authors detected amplification of N-myc in three of eight less differentiated ganglioneuroblastomas in the treated group and observed the worst prognosis in these three patients. The total percentage of the cases from both untreated and treated groups suggest that amplification of N-myc may occur more frequently in undifferentiated types of neuroblastomas than in less malignant types. In conclusion, the amplification of N-myc in neuroblastomas was closely associated with the worst prognosis, which was suggested by both disease stage and histologic characteristics.

Child↗