Biomedical subjects

S Hara

Publications and source records attributed to S Hara.

At least 217 records · Page 12Linked to original sources

PubMed1997

Renovascular hypertension may cause nephrotic range proteinuria and focal glomerulosclerosis in contralateral kidney.

Little attention has been paid to nephropathies and proteinuria in renovascular hypertension (RVH). Recently there has been a growing interest in the conditions induced by RVH. 10 cases of RVH were diagnosed by angiography and renin sampling from renal veins in the last 6 years in our hospital. The patients were all male and mean age was 64 +/- 8 (SD) years. Data were as follow: protein excretion was 3.8 +/- 2.2 g/day (> or = 3.5 g/day in 8 patients), sBP 202 +/- 24 mmHg, dBP 113 +/- 17 mmHg, serum renin concentration 64 +/- 45 pg/ml, and ipsilateral/contralateral renal vein renin ratio 3.3 +/- 1.0. RVH was treated by nephrectomy in 3 patients, percutaneous transluminal renal angioplasty (PTA) in 2, and angiotensin converting enzyme inhibitors (ACE-I) administration in 8. Biopsies were performed on contralateral kidney in 4 patients. Focal segmental glomerulosclerosis (FGS) was found in 3 patients, and nephrosclerosis in 1, whereas only nephrosclerosis was found in nephrectomized kidneys in all 3 patients. After nephrectomy, PTA and the treatment by ACE-I, not only blood pressure but also proteinuria was markedly reduced. These findings suggest that severe stenosis of the renal artery led to renal ischemia, which activated renin excretion, to cause glomerular hyperfiltration through vasoconstriction of the efferent arterioles in the contralateral kidney. FGS-like lesion thus induced appeared to have caused massive proteinuria.

Aged↗

PubMed1997

[A case of hepatic arterial infusion chemotherapy for multiple liver metastasis from breast cancer].

A 52-year-old woman underwent Auchinclass' operation for breast cancer. The histological type was papillotubular carcinoma. One year and 5 months after operation, multiple liver tumors were found on the CT scan and multiple bone metastasis on MRI. The former were treated by hepatic artery infusion chemotherapy with epirubicin and 5-FU using a subcutaneous implanted pump and the latter by 50 Gy irradiation. The patient began to complain of abdominal pain and discomfort after hepatic artery infusion, so all treatment was discontinued. Six months later the patient died of respiratory failure due to pleural dissemination. No liver mass was detected, and bone metastasis was not changed in section tissues. This suggested that the therapy for a breast cancer patient with distant metastasis must be considered according to the region of recurrence.

Adenocarcinoma↗

PubMed1997

[Reduction in mean cerebral blood flow measurements using 99mTc-ECD-SPECT during normal aging].

Mean cerebral blood flow (mCBF) was measured by SPECT using the 99mTc-ECD-Patlak-Plot method in a selected group of 61 normal non-hospitalized subjects aged 51 to 91 years. The mCBF values showed 48.4 +/- 4.7 ml/100 g/min in aged 50-59 years group, 49.9 +/- 5.9 ml/100 g/min in aged 60-69 years group, 46.4 +/- 6.5 ml/100 g/min in aged 70-79 group, 38.0 +/- 3.7 ml/100 g/min in aged 80-89 years group, 38.9 ml/100 g/min in aged 90-99 years group. There was a statistically significant reduction of mCBF with advancing age (R = -0.41; p = 0.001). Women have significantly higher mCBF values than men up to aged 70 years group. In this study, there was no significant laterality in the mCBF between right and left hemispheres in all decade groups. The history of hypertension, alcohol consumption, and cigarette smoking failed to show significant difference in the mCBF values. The present study shows that normal aging is associated with mCBF reduction.

Aged↗

PubMed1997

Expression of tissue factor and tissue factor pathway inhibitor in rats lungs with lipopolysaccharide-induced disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is a frequent complication of endotoxin shock, and modulation of endothelial cell hemostatic properties has been proposed to play an important role in its onset. We examined the in vivo expression of tissue factor (TF) and TF pathway inhibitor (TFPI) in rat lungs of a lipopolysaccharide (LPS)-induced DIC model. Light and electron microscopic studies showed that fibrin-rich thrombi were present in the pulmonary microvasculature 3 hours after intraperitoneal injection of LPS (7.5 mg/kg) and increased in number at 6 hours. In an immunohistochemical study, an increase in number of monocytes in the microvasculature was observed after LPS injection, and many of these cells (> 90%) were positive for TF antigen. However, no TF expression in endothelial cells was detected. Pulmonary endothelial cells showed positive reaction for TFPI antigen before LPS injection, but TFPI-positive endothelial cells markedly decreased in number after LPS injection. mRNA expression of TF increased and that of TFPI decreased in the lung tissue 3 and 6 hours after LPS injection. High values of TF activity were detected in the lung tissue and plasma, whereas TFPI activities decreased after LPS injection. These results indicate that imbalance between TF and TFPI, overexpression of TF, and underexpression of TFPI in the lung may contribute to thrombus formation in this LPS-induced DIC model.

Animals↗

PubMed1997

[Flow cytometric quantification of numerical chromosome aberrations in non-small cell lung carcinomas using formalin-fixed paraffin-embedded tissue].

Fluorescence in situ hybridization with biotinylated repetitive DNA probe specific for the centromeric region of chromosome 17 (p17H8: Oncor) was applied to suspended nuclei which were isolated by Shutte's method from formalin-fixed paraffin-embedded tissue. The tissues were obtained from surgically resected specimens from nine patients with non-small cell lung carcinoma. The isolated nuclei were prepared with 0.05% pepsin/0.1NHCl for 15 minutes at 37 degrees C. Subsequently, these were immersed in 70% acetic acid for 10 seconds at room temperature. After heat denature with hybridization mixture which contained 3 mu 1 DNA probe for 10 minutesat 70 degrees C, 1 x 10(6) nuclei were incubated overnight at 37 degrees C. After washing with 60% formamide/2 x SSC, the hybridized probes were labeled by FITC conjugated avidin. A number of centromeric signals of chromosome 17 wasevaluated by fluorescence microscopy (BH-2, Olympus). Furthermore, a probe-related FITC intensity was quantified using flow cytometry (FACScan, Becton Dickinson). As the results, there was good correlation between a relative fluorescence intensity determined by flow cytometry and a relative fluorescence signal by fluorescence microscopy (p < 0.05).

Aged↗

PubMed1996

Preventing the generation of artifacts during peptide map analysis of recombinant human insulin-like growth factor-I.

Peptide mapping using proteolytic enzymes is one useful technique to characterize proteins. However, developing an optimized peptide map is empirical. Some proteins are resistant to proteolysis and it is thereby difficult to obtain a good peptide map. In many cases, the protease-to-substrate ratio is the first modifier to improve the peptide map. As a consequence of increasing the amount of protease, some complications such as nonspecific cleavage, disulfide interchange, transpeptidation, and autolysis of the protease itself may occur. Recombinant human insulin-like growth factor-I (r-HuIGF-I) has been shown to generate a transpeptidation product and a nonspecifically cleaved product under the conditions reported in the literature. We describe here the completion of a peptide map using a combination of Asp-N and Glu-C (V8 strain) endoproteinases. No apparent transpeptidation, nonspecific cleavage, and disulfide exchange was observed. In situ digest of r-HuIGF-I on the probe was also analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proved to be a quick method to analyze the sample.

Amino Acid Sequence↗

PubMed1996

Limited proteolysis by chymotrypsin of midkine and inhibition by heparin binding.

When digested with a low concentration of chymotrypsin, midkine (MK) underwent limited proteolysis and produced two fragments with Mr of 11,000 and 6,000 Da. The cleavage site was identified as on the carboxyl side of Phe55. This limited proteolysis was specifically inhibited by heparin, but not by other glycosaminoglycans. Using various heparin-derived oligosaccharides with different chain lengths or chemically desulfated heparin derivatives, it was shown that a minimum of 6 monosaccharide units was necessary for the inhibition, and that sulfonyl groups of the heparin disaccharide unit were required for inhibition. The present study showed that MK consists of two domains, N- and C-domains, and that Phe55 localized to the hinge region is exposed on the surface of the molecule. It was also suggested that the N-domain may function as a stabilizing domain against proteolytic degradation of the C-domain in the intact molecule.

Amino Acid Sequence↗

PubMed1996

Local changes in oxygen tension and blood flow in the brain under hyperthermia induced by intracerebroventricular NMDA in rats.

Intracerebroventricular administration of N-methyl-D-aspartate (NMDA; 100 nmol) to rats increased oxygen tension and blood flow of the caudate putamen and the ventral hippocampus. The regional differences in the increase in oxygen tension could not be explained in terms of those in the blood flow. NMDA also increased the temperature of the two brain regions to a similar extent, and these increases preceded that in the rectum. These findings suggest that NMDA receptor stimulation leads to exposure of the brain to a higher oxygen level and higher temperature, which might be involved in NMDA neurotoxicity.

Animals↗

PubMed1996

Disulfide structure and N-glycosylation sites of an extracellular domain of granulocyte-colony stimulating factor receptor.

An extracellular domain containing 603 amino acid residues of human granulocyte-colony stimulating factor receptor was expressed in Chinese hamster ovary cells. The affinity-purified material has previously been shown to dimerize when combined with the ligand. In this paper we have characterized the primary structure of this active receptor. Laser desorption mass spectrometry of the purified receptor showed a broad peak at a molecular weight of 84,000, ranging from 77,000 to 91,000. The molecular weight heterogeneity is due to glycosylation. Since the molecular weight based on the amino acid sequence is 67,322, by subtraction the carbohydrate content is approximately 17,000. Disulfide structure of the receptor was determined by peptide mapping in the absence and presence of reducing agent. Sequence and mass spectral analyses of these peptides showed the receptor to contain eight disulfide bonds and three free cysteines. These disulfide bonds are consistent with the known domain motifs of the receptor in that no interdomain disulfides were present. One of the three free cysteines is reactive with alkylating agents, while the others are less reactive, probably being buried in the interior of the molecule. Blocking the free cysteines did not affect the ligand binding. Carbohydrate moieties are somewhat evenly spaced throughout the molecule, at eight different N-glycosylation sites, some of which show heterogeneity in their compositions. Glycosylation seems necessary for stabilizing the molecule against disulfide-linked oligomerization of the receptor, indicating that the free cysteine residues become reactive for oxidation and disulfide exchange upon deglycosylation.

Amino Acid Sequence↗

PubMed1996

Human gene encoding prostacyclin synthase (PTGIS): genomic organization, chromosomal localization, and promoter activity.

The prostacyclin synthase gene isolated from human genomic libraries (PTGIS) consists of 10 exons spanning approximately 60 kb. All the splice donor and acceptor sites conform to the GT/AG rule. Genomic Southern blot and fluorescence in situ hybridization analyses revealed that the human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q13. 11-q13.13. The 1.5-kb sequence of the 5'-upstream of the translational initiation site contained both GC-rich and pyrimidine-rich regions and consensus sequences of the transcription factor recognition sites such as Sp1, AP-2, the interferon-gamma response element, GATA, NF-kappaB, the CACCC box, and the glucocorticoid response element. The core binding sequence (GAGACC) of the shear stress responsive element was also found in the 5'-flanking region of the gene. The major product of the primer extension analysis suggested that the transcription of the gene started from the positions around 49 bp upstream of the translational initiation codon. Transient transfection experiments using human aortic and bovine arterial endothelial cells demonstrated that the GC-rich region (positions -145 to -10) possessed a significant promoter activity. The 6-kb downstream sequence of the translational termination codon contained multiple polyadenylation signals, Alu repeat sequences, and the consensus sequence of the primate-repetitive DNA element, MER1. Two sizes of the prostacyclin synthase mRNAs (approximately 6 and 3.3 kb) were detected with the human aorta and lung. RNA blot hybridization analysis using the 3'-untranslated region as probe indicated that the sizes of the 3'-flanking regions were different in the major 6-kb and minor 3.3-kb mRNAs.

Animals↗

PubMed1996

Site-directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys-pocket, and Glu347 and Arg350 of the EXXR motif.

The possible active site Cys441 in the Cys-pocket and Glu347 and Arg350 of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441 Ala, Cys441 Ser, Cys441 His, Glu347 Ala and Arg350 Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1alpha/min per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys441 in the Cys-pocket, and Glu347 and Arg350 of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of a new member of the insect defensin family from a beetle, Allomyrina dichotoma.

A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma. The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins. The new defensin was found to be inducible by bacterial injection. Analysis of the antibacterial spectrum of A. dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family. In addition, A. dichotoma defensin also exhibited antibacterial activity against methicillin-resistant S. aureus (MRSA) isolated from a patient.

Amino Acid Sequence↗

PubMed1996

Production in Escherichia of moricin, a novel type antibacterial peptide from the silkworm, Bombyx mori.

Moricin is a novel type antibacterial peptide recently isolated from the silkworm, Bombyx mori. Two foreign gene expression systems in Escherichia coli were employed to obtain a large amount of the peptide for further characterization. An artificial moricin gene was chemically synthesized and inserted into two expression vectors, pXa1 and pMAL-c2. The recombinant moricin was efficiently produced in E. coli as fusion proteins and released by chemical cleavage with cyanogen bromide or o-iodosobenzoic acid. Eleven milligrams of pure recombinant moricin was obtained from 2 L of E. coli culture. The primary structure and molecular mass of the purified recombinant moricin was the same as those of the natural moricin. In addition, the antibacterial activity of the recombinant moricin against E. coli and Staphylococcus aureus was comparable to that of the natural moricin.

Amino Acid Sequence↗

PubMed1996

Effect of protease inhibitors on early events of apoptosis.

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization of in situ DNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence of in situ DNA strand breaks. DNA stability was estimated by the measure of its sensitivity in situ to denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4',6-diamidino-2- phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of >=50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNA in situ, and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to >=50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.

Animals↗

PubMed1996

Structural characteristics of heparin-line domain required for interaction of midkine with embryonic neurons.

Midkine is a heparin binding growth/differentiation factor with neurite promoting activity. We examined inhibitory activities of various heparin derivatives toward interaction of midkine with neurons and elucidated the structural requirements of the heparin-like domain necessary for the interaction. All of the three sulfate groups in the heparin disaccharide unit (6-O-sulfate, 2-O-sulfate and N-sulfate) were necessary for full inhibitory activity. Among these, the N-sulfate group was critically important. The minimum size with inhibitory activity was approximately 7,000 Da. Thus, the highly sulfated region in cell surface heparan sulfate proteoglycan is required for neurons to interact with midkine.

Animals↗

PubMed1996

Hormonal regulation of aldose reductase in rat ovary during the estrous cycle.

The physiological roles of aldose reductase [alditol:NAD(P)+1-oxidoreductase] have not been fully elucidated yet, although it has been implicated in the pathogenesis of diabetic complications. In the rat ovary we found remarkable changes in the enzyme level during the 4-day estrous cycle. After diestrus, the activity and protein content of aldose reductase increased to the maximum level on proestrous morning and rapidly fell off to the lowest level on the early morning of estrus. At this time its mRNA level in the ovary was significantly decreased compared with that on the morning of proestrus. Immunohistochemical staining of the diestrous ovary demonstrated localization of the enzyme protein in the granulosa cells and in the oocytes. At the end of proestrus when its level was low, immunoreactive aldose reductase in the granulosa cells was localized preferentially to the antrum side, with lesser staining in the cells lining the follicles. Administration of chlorpromazine to the rats on proestrus significantly restored the enzyme level on the following morning of the expected estrus. This effect of chlorpromazine was abolished when human chorionic gonadotropin was administered to the chlorpromazine-treated rats. When chlorpromazine was administered to the rats treated with bromocriptine, an inhibitor of pituitary prolactin secretion, aldose reductase activity in the ovary was significantly elevated compared with that in the rats treated with chlorpromazine alone. These findings suggest that in the rat ovary it is under hormonal regulation during the estrous cycle. The enzyme may possess a new functional role in the reproductive system of the female rat, which can be disordered under diabetic conditions.

Aldehyde Reductase↗

PubMed1996

Isolation and structural characterization of recombinant human neu differentiation factor expressed in Escherichia coli.

Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively.

Amino Acid Sequence↗

PubMed1996

Distinct effects of N omega-nitro-L-arginine on seizures induced by several drugs in mice.

A potent nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine (L-NA), suppressed tonic seizure elicited by pentylenetetrazol (PTZ; 100 mg/kg, SC) in a dose-related manner (25 to 100 mg/kg, IP), but had no effect on clonic seizure. The effect was most potent at 1 h after the administration of L-NA. L-NA (100 mg/kg, IP) suppressed clonic seizure as well as tonic seizure in bicuculline-treated (3.0 or 4.5 mg/kg, SC) mice. However, it did not affect seizures elicited by picrotoxin (2.0 to 6.0 mg/kg, SC). On the other hand, N-methyl-DL-aspartate (NMDLA; 300 mg/kg or 350 mg/kg, IP) induced clonic seizure, but tonic seizure was not always noted. All mice with clonic and tonic seizures died, and some mice with clonic seizure died without accompanying tonic seizure. L-NA did not influence NMDLA-induced seizures, but it appeared to enhance NMDLA lethality, though without statistical significance. These findings suggest distinct roles of NO in seizures induced by different drugs in mice.

Animals↗