Genes influencing selective fertilization in Neurospora crassa.

The mutual attraction of conidia to protoperithecia of the opposite mating type was studied genetically in crosses where a mixture of conidia from two different strains, one of which was marked by an ascospore color mutant gene tan spore (ts), was applied to protoperithecia. Selective fertilization was measured as the frequency of perithecia fertilized by conidia from one strain in competition with conidia from another strain. Selective fertilization by a given strain varied throughout the range from 10 to 97% according to the strains of protoperithecial parent. The selective fertilization was revealed to be under the control of two or more loci, which appeared to have multiplicative action. No indication of a cytoplasmic effect on selective fertilization was obtained. The strength of the mutual attraction between conidia and protoperithecia decreased as genetic similarity increased.

Point mutation (-69 G-->A) in the promoter region of cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) from HDL to apolipoprotein (apo) B-containing lipoproteins and plays a crucial role in reverse cholesterol transport, which is a major protective system against atherosclerosis. Genetic CETP deficiency is the most common cause of a marked hyperalphalipoproteinemia (HALP) in the Japanese, and various mutations have been identified in the coding region as well as in the exon/intron boundaries in the CETP gene. In the present study, we identified a novel mutation in the promoter region of the CETP gene. This mutation was a G-to-A substitution at the -69 nucleotide of the promoter region (-69 G-->A), corresponding to the second nucleotide of the PEA3/ETS binding site (CGGAA) located upstream of the putative TATA box. Four (2.0%) of 196 unrelated subjects with a marked HALP (HDL cholesterol >/=2.59 mmol/L=100 mg/dL) were revealed to be heterozygous for the -69 G-->A mutation, and the allelic frequency of the mutant was 0.0102 in the subjects with a marked HALP. The subjects with the -69 G-->A mutation had low plasma CETP levels. Reporter gene assay showed that this mutation markedly reduced the transcriptional activities in HepG2 cells (8% of wild type). These results suggested that this mutation would be dominant negative. In conclusion, a novel -69 G-->A mutation in the CETP gene causes the decreased transcriptional activity leading to HALP.

Astringency of bovine milk whey protein.

Whey protein solutions at pH 3.5 elicited an astringent taste sensation. The astringency of whey protein isolate (WPI), the process whey protein (PWP) that was prepared by heating WPI at pH 7.0, and the process whey protein prepared at pH 3.5 (aPWP) were adjusted to pH 3.5 and evaluated by 2 sensory analyses (the threshold method and the scalar scoring method) and an instrumental analysis (taste sensor method). The taste-stimulating effects of bovine and porcine gelatin were also evaluated. The threshold value of astringency of WPI, PWP, and aPWP was 1.5, 1.0, and 0.7 mg/mL, respectively, whereas the gelatins did not give definite astringency. It was confirmed by the scalar scoring method that the astringency of these proteins increased with the increase in protein concentration, and these proteins elicited strong astringency at 10 mg/mL under acidic conditions. On the other hand, the astringency was not elicited at pH 3.5 by 2 types of gelatin. A taste sensor gave specific values for whey proteins at pH 3.5, which corresponded well to those obtained by the sensory analysis. Elicitation of astringency induced by whey protein under acidic conditions would be caused by aggregation and precipitation of protein molecules in the mouth.

Lipo-PGE1, prostaglandin E1 incorporated in lipid microspheres, protects injury of the liver caused by warm ischemia reperfusion.

Effects of Lipo-PGE1, prostaglandin E1 incorporated in lipid microspheres on liver injury caused by ischemia reperfusion were investigated. Lipo-PGE1 (10 micrograms/kg or 3 micrograms/kg) or vehicle was gradually injected twice via portal vein 5 min prior to induction of ischemia and reperfusion. Rats died within 2 d after liver ischemia of 90 min from the group receiving injection of vehicle alone. Lipo-PGE1 had its most profound effect on the survival of animals subjected to liver ischemia followed by reperfusion when given in two doses, one prior to ischemia, and another prior to reperfusion. Lipo-PGE1 markedly suppressed both the increases in plasma PCOOH (phosphatidyl-choline hydroperoxide) levels and the leakage of GOT, GPT, and LDH from the liver during the ischemia reperfusion. These findings suggest that Lipo-PGE1 may have therapeutic applications in treatment of hepatic injury.

Ethylene oxide polyneuropathy.

Sensorimotor polyneuropathy developed in two workers who had been exposed to ethylene oxide gas repeatedly for several months. Sural nerve biopsies revealed axonal degeneration with mild changes of the myelin sheath. Unmyelinated fibers were also involved. Muscle biopsies showed typical denervation atrophy. Symptoms improved after exposure to ethylene oxide terminated.

Intracerebroventricular administration of endothelin-1 impairs the habituation of rats to a novel environment in conjunction with brain serotonergic activation.

The effects of i.c.v. administration of endothelin-1, at a low dose that does not produce abnormal behaviors such as barrel-rolling, on the emotional state of rats exposed to a novel environment were examined. Changes in the emotional state of rats with a novel environment were evaluated in terms of changes in exploratory activity in the hole-board apparatus, i.e., locomotor activity as well as the number and duration of rearing and head-dipping behaviors. Rats treated with i.c.v. saline showed marked exploratory behaviors immediately after exposure to the hole-board apparatus, but these exploratory behaviors decreased rapidly with time. On the other hand, the habituation of rats to a novel environment was prolonged by the i.c.v. administration of endothelin-1 (0.3 and 1 pmol). Furthermore, we also found that i.c.v. administration of endothelin-1 (1 pmol) significantly increased the serotonin (5-hydroxytryptamine) turnover in some brain regions, i.e., the cerebral cortex, hippocampus and midbrain, and the inhibition of brain 5-hydroxytryptamine synthesis by treatment with p-chlorophenylalanine (200 mg/kg/day, s.c.) for 2 days suppressed the behavioral effects of endothelin-1 (1 pmol, i.c.v.). In addition, i.c.v. administration of endothelin-1 (1 pmol) did not affect the spontaneous motor activity of rats. The present study demonstrated that i.c.v. administration of low doses of endothelin-1 impairs the habituation of rats to a novel environment in conjunction with brain 5-hydroxytryptaminergic activation. These results suggest that the central endothelin system may play a significant role in mediating emotionality.

Changes in monoamine oxidase activity in mouse brain associated with d-methamphetamine dependence and withdrawal.

The long-term effects of d-methamphetamine (MP, 5 mg/kg) on brain mitochondrial monoamine oxidase (MAO) activity were studied in mice given MP intraperitoneally daily for 4 weeks. The MAO activities decreased when serotonin (5-HT) and dopamine (DA) were used as substrates. A marked elevation in MAO activity was seen during MP withdrawal when 5-HT, DA, beta-phenylethylamine (beta-PEA) and norepinephrine (NE) were used as substrates. The kinetics of MAO showed a significant decrease in Km values, but no significant change in Vmax values during MP withdrawal, despite the presence of NE. The Km and Vmax values increased when NE was the substrate. Inhibition of MAO by MP or its metabolites (amphetamine, p-hydroxyamphetamine and p-hydroxymethamphetamine) increased with the use of the following substrates in the order: DA, 5-HT, NE and beta-PEA.

Isoelectric focusing of isoenzymes of monkey platelet monoamine oxidase.

Monkey platelet monoamine oxidase (MAO) was preferentially found as the B-form of the enzyme as observed from differences in substrate specificities, as well as liver MAO. The isoelectric points and molecular weights of platelet MAO subunits were compared with those of monkey liver using sodium dodecyl sulfate-disc polyacrylamide gel electrophoresis and isoelectric focusing-disc gel electrophoresis. The pI value of monkey liver was a single peak at 6.5, but the pI values of monkey platelets were triple peaks at 5.5, 6.5 and 7.0. The molecular weight of MAO subunits in monkey platelets was similar to that of liver, and was found to be about 60,000. These results indicate that MAO-B of monkey platelets differs from MAO-B of the liver, and that it has different electrophoretic properties.

Modulation of neuronal MAO activity, 5-HT uptake and imipramine binding by endogenous substances in dog cerebrospinal fluid.

Addition of small amounts of dog cerebrospinal fluid (CSF) inhibited both type A and type B monoamine oxidase (MAO) in dog brain mitochondria. The inhibition was competitive with 5-HT as substrate, but non-competitive with beta-phenylethylamine as substrate. Tricyclic antidepressants also exhibited competitive inhibition with type A MAO, but were non-competitive with type B MAO. The endogenous materials in CSF activate [3H]-imipramine specific, dose-dependent binding in dog brain preparations. The maximum number of binding sites (Bmax) increased, but the dissociation constant (Kd) was altered significantly in the presence of CSF. Addition of CSF induced a marked activation of uncompetitive [14C]-5-HT uptake in dog brain preparations. Moreover, there were reversibilities of the inhibition of MAO activity or of the activation of imipramine binding and 5-HT uptake by CSF substance after dilution experiment. These results indicate the possible presence of an endogenous psychotic drug-like substance in CSF.

Reversible inhibition of aromatic hydroxylation of methamphetamine in rat liver microsomal preparations pretreated with methamphetamine.

The effects of a single and repeated administration of methamphetamine (MP) in vivo in rats on its own metabolism in vitro were investigated. In both cases, the p-hydroxylation of MP to p-hydroxymethamphetamine by a microsomal fraction from rat liver was inhibited for a period of 16 hr after the last injection of MP. This inhibition was diminished by dialysis of the microsomal preparations. In contrast, the reduced level of cytochrome P-450 in hepatic microsomes from rats pretreated with the SKF 525-A did not revert to the control value after dialysis. When microsomes were preincubated with N-hydroxymethamphetamine, which is the metabolite of MP and a potent substrate for the formation of a metabolic intermediate (MI) complex with cytochrome P-450, the content of the MI was increased and the MP-hydroxylation activity decreased in direct proportion to the length of the preincubation. These results suggest that the inhibition of MP-hydroxylation may be due to reduction of the level of cytochrome P-450 that accompanies the formation of the MI complex. Furthermore, it appears that the complex can be dissociated by dialysis.

Chemiluminescence-HPLC assay of phosphatidylcholine hydroperoxide generated by ischemia-reperfusion in the liver of rats.

To determine cellular damage due to "oxidative stress", we developed a sensitive and specific quantitative assay for phosphatidylcholine hydroperoxide (PCOOH) by coupling HPLC with detection of chemiluminescence (CL). The qualitative and quantitative detection limits of PCOOH by this assay were 0.5 and 2 pmol (based on active oxygen from hydroperoxide). Using this CL-HPLC method, we determined PCOOH levels caused by ischemia-reperfusion in rat livers. The PCOOH levels in livers of control, sham-operated and operated rats with only ischemic treatment were approximately 2 nmol/g wet liver weight. The PCOOH level and several serum parameters of liver injury increased with an increase in the duration of ischemia, and also increased in proportion to the duration of reperfusion. The determination of PCOOH in liver caused by ischemia-reperfusion could be a useful method for investigating liver damage induced by free radicals.

Effects of anti-free radical interventions on phosphatidylcholine hydroperoxide in plasma after ischemia-reperfusion in the liver of rats.

The present study set out to investigate whether plasma phosphatidylcholine hydroperoxide (PCOOH) levels could accurately reflect lipid peroxidation linking to liver damage due to ischemia--reperfusion. PCOOH is a primary peroxidative product of phosphatidylcholine (PC), which is the most important functional lipid in the hepatocellular membrane, and may mediate oxidative stress. We quantified PCOOH and PC in the plasma and liver of rats subjected to hepatic ischemia-reperfusion by chemiluminescence detecting HPLC (CL-HPLC) method. Plasma PCOOH levels showed no significant rise in either the ischemia only group or in the sham-operation group, compared to controls (0.7 nmol/mL plasma). At 60 min subsequent to reperfusion, the PCOOH levels in plasma and liver, as well as the levels of several serum markers of liver injury [lactic dehydrogenase (LDH), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT)] increased in proportion to the duration of ischemia (up to 60 min). During periods of reperfusion following 30 min of ischemia, plasma PCOOH increased biphasically (2 nmol/mL; 12-24 hr duration of reperfusion), and generally ran parallel to that in the liver after more than 60 min of reperfusion. Dose-dependent protective effects against warm ischemia (30 min)-reperfusion (12 hr) injury were clearly demonstrated in the groups treated with allopurinol, diclofenac Na, ascorbic acid (V.C), alpha-tocopherol and coenzyme Q10, but not in those treated with r-h-superoxide dismutase or betamethasone. The rises in plasma PCOOH and serum GOT, GPT and LDH of the ischemia-reperfused rats were ameliorated most in the group pretreated with diclofenac Na, and next most in the group pretreated with V.C. These results indicate that the plasma PCOOH levels are a useful index both for liver cell damage induced by oxygen free radicals generated during ischemia-reperfusion, and to investigate the efficacy of drugs against oxidative stress.

Preferential inhibition of the B-form of monoamine oxidase in the liver of rats given 3'-methyl-4-dimethylaminoazobenzene in the diet.

A and B-form monoamine oxidase (MAO) activities were measured in the liver of rats maintained with a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). A-form MAO activity was similar to the control value throughout the feeding periods with serotonin as substrate. In contrast, B-form MAO activity decreased rapidly and the level of MAO activity was maintained at about 30% with beta-phenylethylamine (beta-PEA) as substrate. 3'-Me-DAB feeding did not cause any changes in MAO activity in the brain of rats. A single administration of 3'-Me-DAB (100 mg/kg p.o.) failed to alter A and B-form MAO activities for up to 4 days after its administration. The mechanism of inhibition of B-form MAO activity in rat liver mitochondria by 3'-Me-DAB was investigated. The inhibition of 3'-Me-DAB of B-form MAO activity, in vitro, was competitive and reversible. There was no difference in the apparent Michaelis constant toward beta-PEA between control and 3'-Me-DAB fed rats. B-form MAO in rat liver mitochondria was titrated with (-)deprenyl; this compound is selective to and an irreversible inhibitor of B-form MAO. The content of B-form MAO in liver mitochondria of rats fed 3'-Me-DAB for 3 weeks was decreased to about 60% of the control level.

Endogenous monoamine oxidase inhibitor-like substances in monkey brain.

The extraction and partial purification of endogenous "monoamine oxidase (MAO) inhibitor-like" material from the monkey brain are described. The endogenous material (F-1 and F-2) obtained after Bio-Gel P-2 gel filtration and silica column chromatography inhibited MAO in the monkey brain mitochondria toward 5-hydroxytryptamine (5-HT), beta-phenylethylamine (beta-PEA), tyramine and dopamine as substrates. The inhibitory effects of F-1 and F-2 were non-linear concentration dependent, and F-1 non-competitively inhibited A-form MAO, while F-2 inhibited A-form MAO competitively and inhibited B-form MAO non-competitively. These substances were more potent inhibitors of A-form than of B-form MAO. F-2 was heat stable but liable to the treatment with pepsin and trypsin. F-1 was not inactivated by heat treatment and digestion with pepsin and trypsin. F-1 may be a low molecular weight (less than 1350) compound, including certain monoamines or their metabolites or other unidentified compounds, while F-2 was a low molecular weight (about 2500) peptide.

Age-related changes in alpha-tocopherol dynamics with relation to lipid hydroperoxide content and fluidity of rat erythrocyte membrane.

Age-related changes in alpha-tocopherol dynamics in plasma and erythrocyte membranes of 10- to 120-week-old rats were investigated by high-performance liquid chromatography (HPLC) with redox detection mode. Furthermore, changes in lipid hydroperoxide content and fluidity of erythrocyte membrane with age were assessed using chemiluminescence-HPLC and Fourier transform infrared spectrophotometer, respectively. A slight increase in the alpha-tocopherolquinone/alpha-tocopherol ratio in erythrocyte membrane and a decrease in the alpha-tocopherol in erythrocyte membrane/alpha-tocopherol in plasma ratio were observed. A significant increase in lipid hydroperoxide content and a marked decrease in the fluidity of erythrocyte membrane were seen with age. These findings suggest that alpha-tocopherol uptake in erythrocyte membrane declines, and utilization rate of alpha-tocopherol in erythrocyte membrane increases age-dependently. These changes, which enhanced lipid peroxidation and consequently reduced membrane fluidity, may be caused by the impairment of this transfer mechanism.

Changes of oligosaccharides and fatty acids in monkey hippocampus by synaptic potentiation.

We measured the release of free fatty acids and structural changes of glycoprotein glycans induced by tetraethylammonium (TEA) salt in hippocampal slices of cynomolgus monkey brain. The release of free fatty acids in the hippocampal slices occurred after synaptic potentiation by TEA in a different manner from rat hippocampus. Arachidonic acid release in monkey hippocampus occurred much faster than that in rat. Several types of glycans of monkey hippocampal glycoproteins were determined depending on the duration time after TEA treatment. 5-Mannose was increased within 2 min, while polysialoglycans were increased after 5 min or later. Comparative study of glycans of monkey and rat hippocampal slices revealed the presence of relatively larger amount of sialo- and multi-anntenary glycans in rat than in monkey. These results indicate that the depolarizing stimulation of monkey hippocampal slices induced the change of glycoprotein glycan structures and release of free fatty acids in a different manner from rat hippocampus.

Effect of vagal autotransplantation and bifemelane hydrochloride on cholinergic markers and event-related potentials in rats with lesions of the nucleus basalis magnocellularis.

In rats lesioned by injecting the ibotenic acid (8 micrograms/site) into the unilateral nucleus basalis magnocellularis (NBM), the effect of treatment with bifemelane hydrochloride (BIF) or autotransplantation of the vagal nodosal ganglion was studied electrophysiologically by serial measurement of the event-related potential (ERP, P300) for 4 weeks. In addition, the effects on cholinergic markers were assessed by determining the specific binding of [3H]QNB (quinuclidinyl benzilate) to the muscarinic acetylcholine receptor (mAChR) as well as the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). The P300 latency was delayed and its amplitude remained low for 4 weeks in NBM-lesioned rats. In contrast, a return to normal occurred after 2-3 weeks in rats given daily intraperitoneal injections of BIF (15 mg/kg) and in autotransplanted rats. In lesioned rats, the cortical ChAT and AChE activities on the affected side did not recover, but the postsynaptic receptor response was transiently activated soon after lesioning. BIF increased specific mAChR binding (an early increase of affinity and a subsequent increase of receptor density) as well as presynaptic ChAT activity. Transplantation achieved the early activation of mAChR binding (increased receptor density) and continuously increased ChAT activity. Thus, the postsynaptic compensatory receptor mechanism of denervation supersensitivity acted as an early response to the depression of presynaptic cholinergic activity, but it could not improve the P300 response until the subsequent increase of cortical ChAT activity. Improvement of P300 combined with cortical cholinergic recovery after nodosal ganglion grafting or administration of BIF suggests that the neocortical ACh level may play an important role in regulating ERP.

Effect of endotoxin-induced reactive oxygen species on sperm motility.

OBJECTIVE: To define the mechanism of infection-induced damage of sperm. DESIGN: The effect of lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) on sperm motility and its modification by scavengers were investigated. SETTING: Research laboratory of a university hospital. PATIENT(S): Normozoospermic semen samples were obtained from 37 healthy volunteers. INTERVENTION(S): The sperms were incubated in the presence of LPS with or without scavengers. MAIN OUTCOME MEASURE(S): Sperm motility was evaluated by a sperm quality analyzer (SQAIIB). ROS formation in semen samples was measured by a Berthold luminometer (LB953). RESULT(S): Motility of spermatozoa was decreased in the LPS-treated samples compared with that in the control groups. ROS was significantly higher in the LPS-treated groups than in the control groups. The addition of ROS scavengers restored the motility index and suppressed ROS production in the LPS-treated semen samples. CONCLUSION(S): These data suggest that endotoxin-induced excessive production of ROS is responsible for the decrease in sperm motility and that antioxidant therapy may be a therapeutic option for infertile men with bacterial genital tract infection.

Use of evoked responses to measure laser photoradiation tissue effects.

The amount of argon laser irradiation necessary to first produce marked changes in somatosensory evoked potentials when applied to the dorsal columns of cats was used to measure the enhancement of tissue response by fluorescein previously injected intravenously. The mean radiant exposure was roughly 2700 J/cm2 in normal control animals compared to a mean radiant exposure of only about 560 J/cm2 in animals given fluorescein. Thus, the presence of fluorescein in these tissues enhanced the laser's effectiveness by a factor of 5. We suggest the use of evoked responses as a measure of laser photobiological effectiveness.

Inhibition of MAO activity, 3H-imipramine binding, 3H-paroxetine binding and 3H-5-HT uptake by human cerebrospinal fluid.

Addition of small amount of human cerebrospinal fluid (CSF) inhibited both types of MAO in monkey brain mitochondria. The specific binding of 3H-paroxetine decreased remarkably with increasing CSF volumes, while 3H-imipramine binding was slightly inhibited. Scatchard analysis of 3H-paroxetine binding in the presence and absence of CSF indicated that the inhibitory effect was associated with a decreased Bmax without an appreciable change in Kd. Addition of CSF induced an inhibition of uncompetitive 3H-5-HT uptake to monkey cerebral membranes. These results indicate that the materials in human CSF inhibit 3H-paroxetine binding, and modulate the uptake system for 5-HT.