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Biomedical subjects

K Murakami

Publications and source records attributed to K Murakami.

At least 163 records · Page 9Linked to original sources
PubMed1999

Male sterility in transgenic mice expressing activin betaA subunit gene in testis.

Activins and inhibins, which are endocrine regulators of anterior pituitary function, have also been reported to participate in the paracrine and autocrine regulation of reproductive function. To determine the in vivo effects of overexpressed activin/inhibin, we generated transgenic mice carrying the human activin/inhibin betaA subunit mini gene under the regulatory control of the mouse methallothionein promoter. In one of the transgenic line analyzed, the betaA subunit gene was preferentially expressed in the testis. Ectopic and allochronic expression of the betaA gene started at 3 weeks after birth and transgenic male mice became sterile in the ensuing several weeks. Histological analysis revealed testicular degeneration in these mice. The results from this transgenic line strongly support the in vivo activity of activin/inhibin in male reproductive functions.

Animals↗
PubMed1999

Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4.

Arachidonic acid (AA) production by Mortierella alpina 1S-4 was investigated using a 50-L fermentor. In order to optimize the dissolved oxygen (DO) concentration and to investigate the effect of DO on morphology, cultivation was carried out under constant DO at various levels in the range of 3-50 ppm. To maintain a DO concentration above 7 ppm, two methods, i.e., the oxygen-enrichment (OE) method (experimental range, 25-90% oxygen gas supplied) and the pressurization (PR) method (experimental range, 180-380 kPa headspace pressure), were used. As a result, the optimum DO concentration range was found to be 10-15 ppm. In this optimum DO concentration range, the AA yield was enhanced about 1.6-fold compared to that obtained at 7 ppm DO, and there was no difference in the AA productivity between the OE and PR methods. When the DO concentration was maintained at 20-50 ppm using the OE method, the morphology changed from filaments to pellets, and the AA yield decreased drastically because of stress due to the limited mass transfer through the pellet wall. When the DO concentration was maintained at 15-20 ppm using the PR method, the morphology did not change, and the AA yield decreased gradually.

Arachidonic Acid↗
PubMed1999

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.

Animals↗
PubMed1999

(3-substituted benzyl)thiazolidine-2,4-diones as structurally new antihyperglycemic agents.

A series of 3-[(2,4-dioxothiazolidin-5-yl)methyl]benzamide derivatives was prepared as part of a search for antidiabetic agents. A structure-activity relationship study of these compounds led to the identification of 5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[4-(trifluorome thyl)-phenyl]methyl]benzamide (KRP-297) as a candidate drug for the treatment of diabetes mellitus.

Animals↗
PubMed1999

Reduction of depressive-like behavior in mice lacking angiotensinogen.

We made use of targeted disruption of the mouse angiotensinogen (ATN) gene to examine the functional role of the ATN in the central nervous system. Both male and female ATN-deficient mice displayed the reduction of depressive-like behavior in the behavioral despair swim tests and spontaneous locomotor activity diminished. However, both male and female ATN-deficient mice showed no anxiogenic-like or memory-deficit behavior and there was no change in the pain threshold. We propose that endogenous ATN in the central nervous system may regulate the depressant state in the brain.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Vascular injury causes neointimal formation in angiotensin II type 1a receptor knockout mice.

Many studies using small-animal models suggest that angiotensin II (Ang II) plays an important role in neointimal formation after vascular injury. In the present study, we examined whether Ang II type 1 receptor (AT1)-mediated Ang II signaling is indispensable for the development of injury-induced neointimal formation using AT1a knockout (KO) mice. Reverse transcriptase-polymerase chain reaction analysis revealed that AT1 mRNA was not detectable in both uninjured and injured carotid arteries of KO mice, whereas the AT1 gene was expressed in uninjured carotid arteries of wild-type (WT) mice. At 14 days after injury, AT1 mRNA levels were increased by 1.5-fold in injured arteries of WT mice. Although AT2 mRNA was not detectable in uninjured arteries, expression of AT2 gene was induced in both animal groups at 2 weeks after injury. Vascular injury induced neointimal formation in KO mice as well as in WT mice. There were no significant differences between WT and KO mice in the extent of histological findings such as increased cross-sectional areas of the neointima and the media, the number of proliferating smooth muscle cells, and the amount of collagen and fibronectin. Treatment with subpressor doses of Ang II after injury enhanced the growth of neointima in WT mice but not in KO mice. Moreover, treatment with the selective AT1 antagonist CV-11974 before injury significantly decreased the formation of neointima in only WT mice, whereas treatment with the selective AT2 antagonist PD-123319 before injury had no effects in both animal groups. These results suggest that AT1-mediated Ang II signaling is not essential for the development of neointimal formation, although it may modify it.

Analysis of Variance↗
PubMed1999

Neutral glycolipid and ganglioside composition of type-1 and type-2 astrocytes from rat cerebral hemisphere.

We reported previously that the major gangliosides in primary mixed-type astrocyte cultures are GM3 and GD3. To obtain more information regarding the exact distribution of glycosphingolipids in different types of astrocytes, we established a line of type-1 astrocytes that are characterized by a Ran-2 positive, broad flat morphology, and by the absence of binding to A2B5 antibodies. We also purified O-2A progenitor cells by immunopanning and cultured them in the presence of 10% newborn calf serum. They differentiated into type-2 astrocytes that were identified by immunostaining for each of GD3, A2B5, and GFAP. Using these cell cultures, we demonstrate that the major gangliosides were GM3 in type-1 astrocytes and GM3 and GD3 in type-2 astrocytes. In addition, a set of neutral glycolipids was identified based on the HP-TLC migration properties of CMH, CDH, CTH, and Glob, but the component distribution of these glycolipids is related to that of glycolipids of astrocytes. A marked increase in the expression of CTH and Glob was shown in type-2 astrocytes. The amount of neutral glycolipid-sugar was higher in the type-2 astrocytes than in the type-1 astrocytes. These results suggest that the increase in the total glycosphingolipid content and the change in the neutral glycolipid composition produced by type-2 astrocytes may be related to their biological functions and the cellular compositions.

Animals↗
PubMed1999

Reduction of CD4+ and CD8+ T lymphocytes during febrile periods in horses experimentally infected with equine infectious anemia virus.

Three horses were experimentally infected with equine infectious anemia virus (EIAV). All horses were febrile after inoculation with EIAV and then developed chronic symptoms with intermittent fever. The febrile period was characterized by a rise in body temperature with reduced PBL and erythrocyte counts. Flow cytometric analysis showed that the reduced number of lymphocytes was due to significant decreases in CD4+ and CD8+ T cells in the absence of any change in B cell number. At the end of the febrile period the body temperature began to recover and numbers of CD4+ and CD8+ T cells showed a tendency to increase. For CD8+ T cells, this increase continued for several days after the febrile period. B cell number also significantly increased after the febrile period in two out of three horses. The decrease of CD8+ T cells was greater than that of CD4+ T cells. Although the PBL numbers and the CD4/CD8 ratio returned to the level of the preinoculation period, erythrocyte numbers decreased as the body temperature normalized after each intermittent fever. These results suggest that the recurring cycle of fever accompanied with viremia is caused by a reciprocal relationship between EIAV replication and the host immune response. Furthermore, we demonstrate that the lymphocytic response mitigates fever and viremia in EIAV infection despite the absence of virus neutralizing antibody.

Animals↗
PubMed1999

Intermolecular and interdomain interactions of a dynamin-related GTP-binding protein, Dnm1p/Vps1p-like protein.

Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity. Mammalian dynamins constitute the dynamin subfamily. DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p. Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains. Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits. In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions. The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the self-assembly. Analyses using the two-hybrid system and co-immunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2). The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin. Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.

Animals↗
PubMed1999

Colocalization of phospholipase D1 and GTP-binding-defective mutant of ADP-ribosylation factor 6 to endosomes and lysosomes.

Phospholipase D (PLD) is involved in various aspects of cellular function. Two isoforms, PLD1 and PLD2, have been identified. PLD1, which has two splicing variants, is regulated by various factors, including ADP-ribosylation factor (ARF). We here show that both variants of PLD1 are predominantly localized to late endosomes and lysosomes, but not to the Golgi apparatus or endoplasmic reticulum in contrast to earlier studies. Furthermore, PLD1s show significant colocalization with an ARF6 mutant defective in GTP binding. The data suggest that PLD1, under the regulation of ARF6, plays a role in the function of endosomes and lysosomes.

ADP-Ribosylation Factor 6↗
PubMed1999

Human HKR isozyme: organization of the hexokinase I gene, the erythroid-specific promoter, and transcription initiation site.

We previously described a cDNA for the human HKR isozyme, whose sequence is identical to that of the ubiquitous HKI isozyme, except for a unique 5' end sequence. Screening a human genomic library with a DNA fragment containing an erythroid-specific sequence we found one clone including 5' ends for both HKR and HKI genes. The first HKR exon was located 3 kb 5' of the first HKI exon. These results confirmed that HKR is produced from the HKI gene by alternate promoter and splicing. The HKI gene consisted of 19 exons. All exon-intron boundaries are conserved among the genes for hexokinase and glucokinase. The HKI gene length was estimated at over 67 kb. The initiation site for the HKR was identified by primer extension. Its promoter did not have a canonical TATA box, but an inverted GATA at nt -177 (i.e., 36 nt 5' to the transcription initiation site). In the HKR promoter a DNA fragment spanning nt -275 to nt -107 exhibited erythroid-specific activity. However, this was absent in shorter promoter fragments (nt -206 to -107 or nt -229 to -107). The sequence nt -275 to -229, which appeared critical for the erythroid-specific expression of the HKR gene, contained a consensus motif for Sp-1 and GATA, CCAAT, and GGAA motifs. The electrophoretic mobility shift assay (EMSA) suggested erythroid-specific cooperative protein-protein interaction in this region. Deletion of the GATA sequence as well as reaction with a specific antibody identified GATA-1 as one of the interacting proteins.

5' Untranslated Regions↗
PubMed1999

Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy-2'-deoxyguanosine in DNA.

The naturally occurring flavonoids caused strand scission of DNA in the presence of copper ion. Flavonoids such as myricetin, baicalein, and quercetin as well as ascorbic acid cleaved plasmid pBR322 DNA and calf thymus DNA potently. Addition of catalase protected DNA from the strand breaks caused by flavonoids. Treatment of calf thymus DNA with these flavonoids or ascorbate plus copper produced 8-hydroxy-2'-deoxyguanosine. Cuprous ion reduced by flavonoids and ascorbic acid may play a key role in the oxidative cleavage of DNA and the formation of base adduct. Mutagenic and carcinogenic action of flavonoids may be explained by the prooxidant effects of the compounds.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Oral clonidine reduces the requirement of prostaglandin E1 for induced hypotension.

PURPOSE: To determine the effects of preanesthetic oral clonidine on the dose of prostaglandin EI (PGEI) required to produce hypotension during anesthesia. METHOD: Oral placebo, 75 microg or 150 microg clonidine were administered 60 min prior to induction of anesthesia. Anesthesia was maintained with O2:N2O (30:70) and isoflurane 1.0%. After hemodynamic stabilization, an infusion of prostaglandin EI was started (0.05 microg x kg(-1) x min(-1)) and the rate of infusion was adjusted to maintain mean arterial pressure (MAP) between 60-70 mm Hg during operation. RESULTS: Duration of hypotension in placebo, 75 microg and 150 microg preanesthetic oral clonidine treated groups were 132+/-46, 117+/-37 and 129+/-56 min, respectively. The PGEI requirement in each group were 1563+/-180 (28.6+/-3.2), 594+/-197 (10.8+/-3.6) and 283+/-30 (5.5+/-3.6) microg (microg x kg(-1)), respectively. In addition, blood loss in each group were 1461+/-389, 805+/-240 and 931+/-40 ml, respectively. CONCLUSION: Preanesthetic oral clonidine decreased the dose of PGEI required to produce hypotension, and decreased the blood loss during operation.

Administration, Oral↗
PubMed1999

Mammary lymphoscintigraphy with various radiopharmaceuticals in breast cancer.

Sentinel node biopsy (SNB) in breast cancer is a promising surgical technique that avoids unnecessary axillary lymph node dissection. To optimize lymphatic mapping with radiopharmaceuticals, mammary lymphoscintigraphy with 30-50 MBq of technetium-99m-diethylenetriamine pentaacetic acid human serum albumin (99mTc-HSAD), technetium-99m-human serum albumin (99mTc-HSA), or technetium-99m-tin colloid (99mTc-TC) were investigated in 69 cases of primary breast cancer. Dynamic early images were obtained during the first 30 or 40 minutes, and static delayed images were obtained 6 hours after tracer injection. Hot spots as sentinel lymph nodes (SLNs) appeared in 51 of 69 cases (74%): in early images in 27 cases and in delayed images in 24 cases. SLNs were visualized more frequently in 23 of the 26 cases (88%) treated with 99mTc-HSAD and in 21 of the 24 cases (88%) treated with 99mTc-HSA than in only 7 of the 19 cases (37%) treated with 99mTc-TC. In 26 of the 51 cases, SLNs were identified as faint spots in delayed images. There was a significant difference in the first appearance of SLNs on the lymphoscintiscan between 43 cases of dense breast parenchyma and 26 cases of fatty breast parenchyma. These results suggest that 99mTc-HSAD or 99mTc-HSA is acceptable for lymphatic mapping, but in cases which have faint spots in delayed images or fatty breast parenchyma, gamma probe-guided SNB may result in failure or misleading false-negative SLNs.

Adult↗
PubMed1999

Developmental stage-specific involvement of angiotensin in murine nephrogenesis.

Angiotensinogen-deleted mice (Agt-KO) show phenotypes of hypotension and renal atrophy. To investigate whether an alternative pathway other than angiotensin II (AII), i.e., processed angiotensin fragments, may play a biological role in nephrogenesis, we analyzed a congenic line of Agt-KO fetuses and neonates derived from two sources: one (Agt-KO/He) from mating with heterozygous angiotensinogen-deleted mice and the other (Agt-KO/Ho) from mating homozygous angiotensinogen-deleted mice. Although Agt-KO/He did not show a typical phenotype at birth, these mice showed papillary atrophy 2 weeks later and thereafter, a marked increase in renal size, i.e., pelvic dilatation. In contrast, Agt-KO/Ho showed renal abnormalities at birth and subsequently died. TUNEL staining and electron microscopy revealed that accelerated papillary apoptosis was present at birth in Agt-KO/Ho and caused abnormal papillary development; however, apoptosis was not detected in Agt-KO/He, suggesting that different mechanisms for the abnormal renal development exist in Agt-KO/He and Agt-KO/Ho. Two-week administration of an angiotensin fragment (3-8), angiotensin IV (AIV), to Agt-KO/He markedly attenuated the renal atrophy, decreasing the incidence from 81% to 14%. However, administration of AIV to fetal Agt-KO/Ho through the mother did not decrease the incidence. This is marked contrast to AII, which prevented renal atrophy in both fetal and neonatal periods. It is therefore suggested that AIV is involved in nephrogenesis in a developmental stage-specific manner.

Angiotensin II↗
PubMed1999

Single-molecule imaging of RNA polymerase-DNA interactions in real time.

Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of approximately 0.66 s-1, which was more than severalfold smaller than the rate at other positions. The association rate constant for the slow dissociation sites was 9.2 x 10(2) bp-1 M-1 s-1. The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions. RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA. The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA. The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription.

Bacteriophage lambda↗
PubMed1999

Amelioration by KRP-297, a new thiazolidinedione, of impaired glucose uptake in skeletal muscle from obese insulin-resistant animals.

We examined the effect of KRP-297, a new thiazolidinedione derivative, on glucose uptake in the soleus muscle of two animal models of insulin resistance that show moderate (ob/ob mice) and severe (db/db mice) hyperglycemia. Insulin-stimulated 2-deoxyglucose (2DG) uptake in soleus muscle was 53.8% lower in ob/ob mice versus lean mice (P < .05). When administered to ob/ob mice, KRP-297 (0.3 to 10 mg/kg) decreased plasma glucose and insulin levels and improved the impaired insulin-stimulated 2DG uptake in soleus muscle in a dose-dependent manner. Soleus muscle from db/db mice exhibited defects in both basal (35.0% decrease, P < .01) and insulin-stimulated (50.5% decrease, P < .01) 2DG uptake. These defects were improved by treatment with KRP-297 (0.3 to 10 mg/kg). Moreover, KRP-297 prevented severe hyperglycemia and the marked decrease in pancreatic insulin content in db/db mice. These results suggest that KRP-297 treatment is useful to prevent the development of diabetic syndromes in addition to ameliorating the impaired glucose transport in skeletal muscle.

Animals↗