Biomedical subjects

M Nishihara

Publications and source records attributed to M Nishihara.

At least 91 records · Page 5Linked to original sources

PubMed1997

Purification and properties of sn-glycerol-1-phosphate dehydrogenase from Methanobacterium thermoautotrophicum: characterization of the biosynthetic enzyme for the enantiomeric glycerophosphate backbone of ether polar lipids of Archaea.

The enzyme which seems to be responsible for the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids was purified from a methanogenic archaeon, Methanobacterium thermoautotrophicum, and characterized. The enzyme, sn-glycerol-1-phosphate: NAD(P)+ oxidoreductase (sn-glycerol-1-phosphate dehydrogenase), was purified 7,600-fold from a cell free extract by ammonium sulfate fractionation and seven steps of chromatography. The final preparation exhibited a specific activity of 617 micromol/min/mg (Vmax) and gave a single band corresponding to 38 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme showed an apparent molecular mass of 302 kDa on gel-filtration chromatography, indicating it is present as a homooctamer. Maximum activity was observed at 75 degrees C at near neutral pH. The activity was stimulated by potassium ions. The Km for dihydroxyacetone phosphate was 7.5 times smaller than that for sn-glycerol-1-phosphate, suggesting that the formation of sn-glycerol-1-phosphate is the natural direction in the cell. Under the assay conditions used, no product inhibition was observed. The N-terminal amino acid sequence was determined.

Amino Acid Sequence↗

PubMed1997

Different female reproductive phenotypes determined by human growth hormone (hGH) levels in hGH-transgenic rats.

The effect of continuous human GH (hGH) secretion on female reproduction was studied in adult female transgenic rats expressing the hGH gene with a mouse whey acidic protein (mWAP) promoter. Two lines of transgenic female rats carrying the mWAP/hGH gene were established and used in the study. One was characterized by relatively high levels of serum hGH (high line), and the other had relatively low levels (low line). High-line female rats had recurring, pseudopregnancy-like estrous cycles accompanied by increased serum progesterone levels for 2 wk after ovulation, and they were fertile. In these rats, luteinization occurred spontaneously without cervical stimulation, probably due to high levels of serum hGH, which has prolactin (PRL)-like activity in the rat. Although low-line female rats had recurring, regular 4-day estrous cycles, they were sterile. In these rats, pseudopregnancy could not be induced by mating or by mechanical cervical stimulation. PRL surges following cervical stimulation were not detected, and PRL secretion was not induced by a dopamine antagonist, metoclopramide. The ovarian tissue in this line had a much higher number of corpora lutea and grew much heavier than in normal littermates, suggesting impairment of PRL-induced structural luteolysis. Suppression of PRL secretion in low-line rats was, at least in part, due to a marked decrease in the number of lactotrophs in the pituitary. The present study shows that the serum hGH level plays a crucial role in regulating luteal function in female transgenic rats expressing the hGH gene.

Animals↗

PubMed1997

A novel steroid inhibitor for a rat 20alpha-hydroxysteroid dehydrogenase isozyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD, E.C.1.1.1.149) in rat luteal tissue, which catalyzes conversion of progesterone to a biologically inactive steroid, 20alpha-hydroxypregn-4-ene-3-one (20alpha-OHP), suppresses progesterone secretion into the circulation. An increase in 20alpha-HSD activity in luteal tissue in rats is a prerequisite for functional corpus luteum regression. This study was undertaken to find a steroid inhibitor for ovarian cytosolic 20alpha-HSD activity among derivatives based on progesterone structure. A derivative designated as STZ26 (D-homo-16-oxa-4-androstene-3,16alpha-dione) was found to inhibit potently 20alpha-HSD activity in cultured luteal cells. Ovarian 20alpha-HSD activity consists of two isoforms (HSD1 and HSD2). Kinetic analyses of STZ26 for HSD1 and HSD2 showed that the compound suppressed only HSD1 activity by competitive inhibition. Pseudopregnant rats were treated with STZ26 from 13 to 19 days after cervical stimulation. Either an elevation of peripheral 20alpha-OHP levels or a concomitant depletion of peripheral progesterone levels at the end of pseudopregnancy was considerably inhibited in treated animals, although not completely. The results showed that STZ26 is a biologically active inhibitor for HSD1 activity in the luteal tissue and suggested that the depletion of progesterone levels toward the end of pseudopregnancy is not solely due to the elevation of HSD1 activity.

20-Hydroxysteroid Dehydrogenases↗

PubMed1997

Production of 5,8,11-Eicosatrienoic Acid (Mead Acid) by a (Delta)6 Desaturation Activity-Enhanced Mutant Derived from a (Delta)12 Desaturase-Defective Mutant of an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4.

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3(omega)9) was attained by a mutant fungus, Mortierella alpina M209-7, derived from (Delta)12 desaturase-defective M. alpina Mut48. The 20:3(omega)9 production by M209-7 was 1.3 times greater than that by its parent strain, Mut48. This is thought to be due to its enhanced (Delta)6 desaturation activity, which was 1.4 times higher than that of Mut48. In both strains, 87 to 88% of the total lipids comprised triacylglycerol (TG) and 85% of 20:3(omega)9 was contained in TG. On optimization of the culture conditions for M209-7, earlier glucose feeding and shifting of the growth temperature from 28 to 19(deg)C on the second day were shown to be effective. Under the optimal conditions with a 10-liter jar fermentor, 20:3(omega)9 production reached 1.65 g/liter of culture medium (corresponding to 118 mg/g of dry mycelia and 28.9% of total fatty acids), which is about twice that reported previously (0.8 g/liter).

Journal Article↗

PubMed1997

On the role of gonadotropin-releasing hormone (GnRH) in the operation of the GnRH pulse generator in the rhesus monkey.

The pulsatile secretion of luteinizing hormone (LH), occasioned by the pulsatile release of gonadotropin-releasing hormone (GnRH), is closely associated with concurrent increases in multiunit electrical activity in the mediobasal hypothalamus (MUA volleys), the electrophysiological correlates of GnRH pulse generator activity. These volleys represent a highly synchronized increase in firing frequency of individual neurons. The origin of these rhythmic oscillations in unit activity and the mechanisms responsible for their synchronization are unknown. The purpose of the present study was to examine the role, if any, of GnRH in the functioning of the GnRH pulse generator in rhesus monkeys. Ovariectomized animals bearing recording electrodes chronically implanted in the mediobasal hypothalamus and fitted with intracerebroventricular (ICV) cannulae in the lateral ventricle and with indwelling cardiac catheters were studied. LH was measured in venous blood withdrawn from the cardiac catheters every 10 min while hypothalamic electrical activity was monitored continuously. In Experiment 1, following a 3- to 4-hour control period, GnRH was infused ICV at a rate of 300 ng/kg body weight (BW)/h over 4-5 h. In Experiment 2, antide, a long-acting GnRH antagonist, was injected ICV in a dose of 105 microg/kg BW after a control period of 3-4 h. Additional control experiments were performed in each animal using vehicle alone. Neither GnRH nor antide affected the frequency of MUA volleys and attendant LH pulses despite significant alterations in LH secretion. These results suggest that, in the rhesus monkey, GnRH may not be involved in the operation of the GnRH pulse generator.

Animals↗

PubMed1997

Prolactin expresses differential effects on apoptotic cell death of luteal cells in vivo and in vitro.

PRL surges in female rats have dual effects of luteal function: either inducing luteolysis during the estrous cycle or rescuing and maintaining luteal function during pseudopregnancy. We analyzed these apparent contradictory effects in relation to apoptosis. The detection of fragmented DNA and in situ 3'-end labeling studies were done on corpora lutea (CL) collected from cycling rats at proestrus 1800 h (P1800 specimen) or pseudopregnant rats on day 6 (psp 6). Distinct DNA ladders were observed in P1800 samples as we previously reported, but only slight ones were found in psp 6 specimen. The effect of PRL on the induction of apoptosis was evaluated in vitro with dispersed luteal tissue. CL from cycling rats were exempted from a PRL surge by pre-treating donors with a dopamine agonist. The extent of apoptotic reaction in P1800 specimen depended on the doses of PRL added to the culture medium. In psp 6 specimen, in contrast, PRL suppressed the apoptotic reaction, increased the cell survival rate (MTT assay), and decreased the cell death rate (LDH assay). Furthermore, PRL enhanced 20 alpha-hydroxysteroid dehydrogenase activity in P1800 specimen but suppressed it in psp 6 specimen. In summary, PRL in rats is either an apoptosis-inducer or -suppressor, depending on the functional state of luteal cells.

Animals↗

PubMed1997

Tumor necrosis factor-alpha mediates endotoxin induced suppression of gonadotropin-releasing hormone pulse generator activity in the rat.

Bacterial endotoxin lipopolysaccharide (LPS) is known to suppress gonadotropin secretion and this effect is assumed to be mediated by cytokines. In the present study, we examined whether LPS affected hypothalamic electrical activity associated with LH pulses, and whether tumor necrosis factor-alpha (TNF-alpha), a major cytokine induced by LPS, was involved in this process. Ovariectomized rats were fitted with chronically implanted electrode arrays in the mediobasal hypothalamus, and multiunit activity (MUA) was recorded under conscious, unrestrained conditions. Blood samples were withdrawn every 6 min through an indwelling atrial catheter or determining serum LH concentrations. Intravenous (i.v.) injection of LPS (1 microgram) suppressed characteristic increases (volleys) in MUA associated with LH pulses throughout the experimental period up to 5 h. This suppressive effect of LPS on MUA volleys was significantly attenuated by simultaneous intracerebroventricular (icv) injection of the antibody (50 ng) to TNF-alpha through an indwelling cannula in the lateral ventricle. These changes in MUA were faithfully reflected in the LH secretory pattern. Further, either i.v. (0.4-2 micrograms) or i.c.v. (20-250 ng) injection of TNF-alpha suppressed the frequency of MUA volleys and associated LH pulses in a dose-dependent manner. These results suggest that LPS leads to the suppression of gonadotropin-releasing hormone pulse generator activity through a mechanism involving TNF-alpha.

Animals↗

PubMed1997

[Efficacy of Warm Touch warming system for hypothermia].

Anesthetized surgical patients frequently become hypothermic, because of decreased metabolic heat production, increased heat loss, surgical exposure, and dry respiration gases. Intraoperative hypothermia may trigger postoperative protein breakdown, shivering, myocardial ischemia, and many other problems. For that reason, heat conservation is a major anesthetic management. We determined the efficacy of Warm Touch warming system (Mallinckrodt Medical, Inc.) compared with that of a warming blanket. Sixteen patients undergoing oral and maxillo-facial surgery under neuroleptanesthesia were studied by measuring rectal and finger-tip skin temperature. Patients were divided in Warm Touch group (n = 8) using Warm Touch warming system Model 5100 and warming blanket group (n = 8) and the temperatures were measured every quarter over 60 minutes. Rectal temperature increased 0.62 degrees C after 60 minutes in the Warm Touch group, but significant changes were absent in warming blanket group. Temperature gradient between the rectum and finger-tip skin decreased markedly in the Warm Touch group. This study suggests that Warm Touch is useful to restore body temperature and to prevent postoperative problems arising from intraoperative hypothermia.

Body Temperature↗

PubMed1996

The rostral ventrolateral medulla mediates suppression of the circulatory system by the ventromedial nucleus of the hypothalamus.

We recently reported that a train of episodic neural discharges within the ventromedial nucleus of the hypothalamus (VMH) associated with suppression of the circulatory system had been determined by monitoring multiple unit activity (MUA). Abrupt increases in neural activity (MUA volleys; 1 to 4 min in duration) accompanied transient decreases in heart rate (HR) and blood pressure (BP), and showed circadian rhythm, occurring every 15 to 30 min in the light phase but seldom in the dark phase. The present study was aimed to determine if neurons in the vasomotor area of the rostral ventrolateral medulla (RVL) are involved in this VMH-induced cardiovascular suppression. MUAs of the VMH and RVL were monitored simultaneously with HR and BP in urethane-anesthetized rats. In synchrony with each MUA volley in the VMH, spontaneous activity of RVL neurons significantly decreased, as well as HR and BP. These RVL neurons are most likely vasomotor neurons because MUA of the RVL was attenuated by baroreceptor reflex activation, and electrical stimulation of these cells through the MUA recording electrodes produced pressor responses. These data suggest that VMH neurons that show a train of episodic discharges suppress the circulatory system at least in part by inhibiting the excitability of vasomotor neurons in the RVL.

Animals↗

PubMed1996

Liver glycogen in fasted rat livers does not improve outcome of liver transplantation.

Controversy exists over how the nutritional condition of the donor liver affects transplant outcome. Some studies suggest that livers from fasted animals (liver glycogen-depleted) are more readily injured than livers from fed animals. Our previous study suggested the opposite, i.e., livers from donors fasted for 4 days were significantly more viable on orthotopic liver transplantation. Fasting may decrease the sensitivity of the liver to an inflammatory response or block Kupffer cell activation following transplantation. Thus, long-term fasting may be beneficial for reasons unrelated to liver glycogen content. In this study we attempted to separate out the roles of fasting and liver glycogen in liver transplant outcome by fasting donors for 2 days and then feeding them only glucose to elevate liver glycogen. Rats (Brown Norway) were fed (standard diet), fasted (4 days), or fasted 2 days and then fed glucose (in water) for 2 days. Livers were preserved for either 30 or 44 h in UW solution and transplanted. Four-day fasting of the donor improved the survival rate in liver transplantation (50%-100% in 30-h cold storage, 29%-83% in 44-h cold storage). However, feeding glucose for 2 days to fasted animals caused a decrease in survival in this series of transplants (40% in 30-h cold storage, 0% in 44-h cold storage). In the glucose-fed group, liver glycogen was 240% of that in the control group. This suggests that the presence of a high concentration of liver glycogen is not beneficial to the preserved and transplanted rat liver.

Animals↗

PubMed1996

Neural activity in the VMH associated with suppression of the circulatory system in rats.

Spontaneous neural activity within the ventromedial nucleus of the hypothalamus (VMH) was monitored in rats to search for neurons regulating the autonomic nervous system. By means of multiple unit activity (MUA) recording method, unique explosive rises in neural activity (MUA volleys), 1 to 4 min in duration, were recorded in conscious freely moving animals. Heart rate was monitored as an autonomic parameter and found to decrease when MUA volleys appeared. These MUA volleys also occurred under urethane anesthesia, and blood pressure and heart rate decreased simultaneously with the volleys, but body temperature remained constant. This fall in blood pressure (but not heart rate) was replicated by electrical stimulation through the electrodes that recorded MUA volleys, suggesting that the neurons responsible for MUA volleys can suppress the circulatory system. The frequency of MUA volleys exhibited a clear diurnal variation: they appeared every 15 or 30 min in the light phase but only seldom in the dark. This diurnal variation seems to be an endogenous circadian rhythm because it was indicated to freerun after blinding the animals. These results suggest that there is a discrete population of neurons in the VMH that fires predominantly during the light phase in an episodic manner and suppresses the circulatory system.

Animals↗

PubMed1996

Changes in responsiveness to serotonin on rat ventromedial hypothalamic neurons after food deprivation.

The effects of food deprivation on responsiveness of neurons in the ventromedial nucleus of the hypothalamus (VMH) to serotonin (5-HT), norepinephrine (NE), gamma-aminobutyric acid (GABA), and neuropeptide Y (NPY) were investigated using brain slices in vitro along with behavioral changes in vivo during fasting. Adult male rats were fasted for 48 h starting at the beginning of the dark phase (lights on: 0700-1900 h). The animals showed a significant loss of body weight on the second day of fasting and an increase in food consumption on the first day of refeeding. During fasting, voluntary locomotor activity was significantly increased in the light phase but not during the dark phase. Plasma catecholamine levels were not affected by fasting. In vitro electrophysiological study showed that, in normally fed rats, 5-HT and NE induced both excitatory and inhibitory responses, while GABA and NPY intensively suppressed unit activity in the VMH. Food deprivation for 48 h significantly changed the responsiveness of VMH neurons to 5-HT, for instance, the ratio of neurons whose activity was facilitated by 5-HT was significantly decreased. The responsiveness of VMH neurons to NE, GABA, and NPY was not affected by food deprivation. These results suggest that food deprivation decreases the facilitatory response of VMH neurons to 5-HT, and that this change in responsiveness to 5-HT is at least partially involved in the increase in food intake motivation and locomotor activity during fasting.

Animals↗

PubMed1996

Occurrence of deoxyribonucleic acid fragmentation during prolactin-induced structural luteolysis in cycling rats.

We determined whether fragmentation of genomic DNA, one of the hallmarks of apoptosis, occurs during structural luteolysis in cycling rats. Corpora lutea (CL) were collected from rats at each estrous cycle stage (1800 h), and fragmented DNA was extracted. Only CL from rats at the proestrous stage showed distinct DNA fragmentation. To determine the period of occurrence of DNA fragmentation, CL were collected at several points between 1200 h on the day of proestrus and 0600 h on the day of estrus. Distinct DNA fragmentation was observed from 1800 h (proestrus) to 2400 h (proestrus), and the extent was significantly lower at 0600 h (estrus). It is known that prolactin (PRL) induces structural luteolysis in rats. To examine the role of PRL in luteal DNA fragmentation, 2-bromo-alpha-ergocryptine (BE) was used to suppress the PRL surge on the day of proestrus. CL collected at 1800 h from BE-treated rats did not show distinct DNA fragmentation, and PRL injection offset the effect of BE. Histochemical analysis with a 3'-end labeling technique confirmed the occurrence of DNA fragmentation in luteal tissue. These results suggest that apoptotic cell death occurs during PRL-induced structural luteolysis.

Animals↗

PubMed1996

Splenic macrophages can modify steroidogenesis of Leydig cells.

A large amount of LH/hCG treatment given to male rats is known to suppress the enzyme activity of cytochrome P450c17 in Leydig cells for 48 h. A high dose LH/hCG injection is also known to allow immunocytes, such as macrophages, to migrate into the testicular interstitial compartment. It has not been known, however, whether these cells play a role in that suppression. In this study, we examined if splenic macrophages have any effects on testosterone secretion from Leydig cells by culturing rat testicular interstitial cells (TIC). Splenic macrophages co-cultured with TIC significantly suppressed testosterone secretion. Macrophages co-cultured reduced both progesterone to testosterone conversion and the amount of cytochrome P450c17 mRNA. The conditioned medium (SMCM), prepared by culturing macrophages for 12 h, significantly reduced either testosterone secretion from TIC or progesterone to testosterone conversion by TIC. These results indicate that splenic macrophages suppress testosterone secretion from Leydig cells by suppressing the cytochrome P450c17 enzyme in vitro, and that this effect is mediated at least in part by some soluble factors secreted from macrophages. Splenic macrophages migrating into the testis after LH/hCG stimulation could play a role in suppressing cytochrome P450c17 in Leydig cells.

Animals↗

PubMed1996