Depletion of the squalene synthase (ERG9) gene does not impair growth of Candida glabrata in mice.

Squalene synthase (farnesyl-diphosphate farnesyltransferase, EC 2.5. 1.21) is the first committed enzyme of the sterol biosynthesis pathway. Inhibitors of this enzyme have been intensively studied as potential antifungal agents. To assess the effect of deactivating squalene synthase on the growth of fungi in mice, we isolated the squalene synthase (ERG9) gene from the pathogenic fungus Candida glabrata and generated strains in which the CgERG9 gene was under the control of the tetracycline-regulatable promoter. Depletion of the ERG9 gene by doxycycline (DOX), a derivative of tetracycline, decreased the cell viability in laboratory media, whereas it did not affect cell growth in mice at all. The growth defect caused by DOX in laboratory media was suppressed by the addition of serum. Analyses of the sterol composition of the restored cells in serum-containing media suggest that the defect of ergosterol biosynthesis can be complemented by the incorporation of exogenous cholesterol into the cells. Thus, deactivation of squalene synthase did not affect fungal growth in mice, presumably because the cells were able to incorporate cholesterol from the serum. These results showed that squalene synthase could not be a suitable target of antifungals for the treatment of C. glabrata infection.

Effect of arrest time and cerebral perfusion pressure during cardiopulmonary resuscitation on cerebral blood flow, metabolism, adenosine triphosphate recovery, and pH in dogs.

OBJECTIVES: To test the hypothesis that greater cerebral perfusion pressure (CPP) is required to restore cerebral blood flow (CBF), oxygen metabolism, adenosine triphosphate (ATP), and intracellular pH (pHi) levels after variable periods of no-flow than to maintain them when cardiopulmonary resuscitation (CPR) is started immediately. DESIGN: Prospective, randomized, comparison of three arrest times and two perfusion pressures during CPR in 24 anesthetized dogs. SETTING: University cerebral resuscitation laboratory. INTERVENTIONS: We used radiolabeled microspheres to determine CBF and magnetic resonance spectroscopy to derive ATP and pHi levels before and during CPR. Ventricular fibrillation was induced, epinephrine administered, and thoracic vest CPR adjusted to provide a CPP of 25 or 35 mm Hg after arrest times of O, 6, or 12 mins. MEASUREMENTS AND MAIN RESULTS: When CPR was started immediately after arrest with a CPP of 25 mm Hg, CBF and ATP were 57 +/- 10% and 64 +/- 14% of prearrest (at 10 mins of CPR). In contrast, CBF and ATP were minimally restored with a CPP at 25 mm Hg after a 6-min arrest time (23 +/- 5%, 16 +/- 5%, respectively). With a CPP of 35 mm Hg, extending the no-flow arrest time from 6 to 12 mins reduced reflow from 71 +/- 11% to 37 +/- 7% of pre-arrest and reduced ATP recovery from 60 +/- 11% to 2 +/- 1% of pre-arrest. After 6- or 12-min arrest times, brainstem blood flow was restored more than supratentorial blood flow, but cerebral pHi was never restored. CONCLUSIONS: A CPP of 25 mm Hg maintains supratentorial blood flow and ATP at 60% to 70% when CPR starts immediately on arrest, but not after a 6-min delay. A higher CPP of 35 mm Hg is required to restore CBF and ATP when CPR is delayed for 6 mins. After a 12-min delay, even the CPP of 35 mm Hg is unable to restore CBF and ATP. Therefore, increasing the arrest time at these perfusion pressures increases the resistance to reflow sufficient to impair restoration of cerebral ATP.

Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae.

We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was abolished. This indicated that derepression of UPR-MS-2 -mediated gene expression was dependent on Snf1p, as is the case of genes encoding isocitrate lyase and gluconeogenic enzymes in S. cerevisiae.

A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3.

We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.

Cerebral blood flow during inhibition of brain nitric oxide synthase activity in normal, hypertensive, and stroke-prone rats.

BACKGROUND AND PURPOSE: Because tonic production of nitric oxide (NO) is important in regulating cerebrovascular tone and NO may be important in the mechanism of brain injury from focal ischemia, we speculated that stroke predisposition in spontaneously hypertensive stroke-prone rats (SHR-SP) may be related to impaired tonic production of NO. This study was designed to test the hypothesis that the cerebral blood flow (CBF) response to inhibition of NO synthase in SHR-SP would be different than that observed in normal Wistar-Kyoto (WKY) rats and non-stroke-prone spontaneously hypertensive rats (SHR). METHODS: Pentobarbital-anesthetized, mechanically ventilated rats were tested for CBF response to saline, 5 or 20 mg/kg IV of NG-monomethyl-L-arginine (L-NMMA), or 20 mg/kg IV of N omega-nitro-L-arginine (L-NA). In addition, specificity for an NO-dependent mechanism was assessed by determining the ability to reverse any alteration in CBF with L-arginine. Hemorrhage was used to minimize any increase in mean arterial blood pressure (MABP) from NO synthase inhibition. In a separate cohort of rats, differential sensitivity of NO synthase for inhibition by nitro-arginine analogues was determined. RESULTS: Baseline MABP was greater in SHR-SP (185 +/- 3, n = 38) and SHR (169 +/- 3, n = 38) compared with WKY rats (101 +/- 2 mm Hg, n = 38, P < .05). Baseline CBF was similar between strains; however, cerebrovascular resistance was higher in SHR-SP (2.16 +/- 0.09, n = 27) and SHR (1.94 +/- 0.07, n = 27) compared with WKY rats (1.23 +/- 0.06 mm Hg/mL per minute per 100 g, n = 27, P < .05). CBF was unchanged with 5 mg/kg L-NMMA or with L-arginine in the absence of L-NMMA in each strain. CBF decreased similarly in SHR and SHR-SP (n = 9 each) in response to 20 mg/kg L-NMMA (SHR, 85 +/- 6 to 67 +/- 6; SHR-SP, 87 +/- 7 to 69 +/- 5 mL/min per 100 g) and was completely reversed by L-arginine. CBF did not decrease with 20 mg/kg L-NMMA in WKY rats. Administration of L-NA (n = 5 each) produced similar reduction of CBF (WKY rats, 67 +/- 6%; SHR, 49 +/- 9%; SHR-SP, 61 +/- 6% of baseline) and inhibition of NO synthase in each strain (approximately 80% inhibition). CONCLUSIONS: There was no difference in the cerebrovascular response to NO synthase inhibition in SHR-SP and non-stroke-prone SHR. Therefore, it is unlikely that an altered sensitivity of NO synthase to inhibition can explain predisposition to stroke in SHR-SP.

Brain nitric oxide synthase activity in normal, hypertensive, and stroke-prone rats.

BACKGROUND AND PURPOSE: Nitric oxide-mediated cerebral vasodilation is altered in spontaneously hypertensive stroke-prone rats. Stroke predisposition in this strain could be related to a genetic defect of brain nitric oxide synthase, the enzyme responsible for nitric oxide production. We tested the hypothesis that brain nitric oxide synthase activity is altered in spontaneously hypertensive stroke-prone rats compared with spontaneously hypertensive or Wistar-Kyoto rats. METHODS: A colony of spontaneously hypertensive stroke-prone rats was bred, in which the rate of neurological events under salt load was assessed. In a separate cohort of animals brain nitric oxide synthase activity was measured in spontaneously hypertensive stroke-prone rats (n = 6) and in spontaneously hypertensive (n = 6) and genetically related Wistar-Kyoto rats (n = 6). Calcium dependency of nitric oxide synthase was also assessed in cortical brain samples from the three rat strains to determine if altered calcium-dependent activation of nitric oxide synthase was present. RESULTS: Brain nitric oxide synthase activity was highest in the cerebellum (eg, spontaneously hypertensive stroke-prone rats: cerebral cortex, 10.6 +/- 0.9; cerebellum, 50.1 +/- 12.0; brain stem, 14.7 +/- 10.3 pmol/mg protein per minute); however, there was no difference among the three rat strains in any region (eg, cerebral cortex: spontaneously hypertensive stroke-prone, 10.6 +/- 0.9; spontaneously hypertensive, 10.8 +/- 0.5; Wistar-Kyoto, 10.9 +/- 0.7 pmol/mg protein per minute) or at any calcium concentration tested. CONCLUSIONS: A genetic defect of brain nitric oxide synthase is unlikely to be the cause of stroke predisposition in spontaneously hypertensive stroke-prone rats.

Rapid monitoring of changes in water diffusion coefficients during reversible ischemia in cat and rat brain.

Changes in the diffusion constant of water during reversible brain ischemia and cardiac arrest were monitored with a 10-s time resolution. Results (five cats, three rats) indicate that these changes are reversible and that the bulk of the changes are not caused by temperature or motion related to brain pulsations and blood flow. The rapid time course of the changes corresponds to the known time course for changes in energy state, signal transduction, and ionic homeostasis.

Regional cerebral blood flow and P3 variables in patients with multiple lacunar infarcts.

We measured regional cerebral blood flow (rCBF) and event-related potentials (ERPs) in 36 patients with multiple lacunar infarcts and 14 age-matched normal subjects. rCBF was measured by the 133Xe inhalation method. ERPs were recorded during visual discrimination tasks using three kinds of stimuli. The patients showed lower mean cortical blood flow than normal subjects especially for the frontal cortex. Nontarget P3 latency in patients was longer than in normal subjects, while no significant differences could be found in target P3 latency between the two groups. Nontarget P3 latency correlated with frontal CBF. These results show that frontal lobe dysfunction may be particularly marked with multiple lacunar infarcts and suggest that reduction of frontal CBF is related to the impairment of the automatic processing associated with the nontarget P3 component.

[Comparison of the indirect immunobead test and the sperm immobilization test for detection of antisperm antibody].

The indirect immunobead test (indirect IBT; IgG.IgA) and the sperm immobilization test (SIT) were carried out for 75 infertile patients to detect antisperm antibodies in the sera. The results were as follows. 1) Twenty three cases showed positive results in the IBT, and 14 out of the 23 showed also positive in the SIT. 2) Fifty two cases which had negative results in IBT also had negative results in SIT. 3) IgG-IB attached to sperm were observed in 14 with positive SIT, but no IgA-IB were observed in 4 cases out of the 14. 4) IgG-IB attached to both the sperm head and end-tail in 12 cases out of the 14, but only to the sperm end-tail in the other 2 cases. We therefore concluded that, 1) IBT detected anti-sperm antibodies more readily than SIT. 2) IBT was an alternative to SIT for screening. 3) Sperm immobilization antibodies appeared to be in the IgG class rather than in the IgA class. 4) It appeared that sperm immobilization antibodies might be able to attach to the sperm tail as well as the head.

[Clinical significance of direct immunobead test to detect anti-sperm antibody].

Direct immunobead test (IBT) (IgG.IgA) was carried out for 290 infertile men to detect antisperm antibodies. The results, compared with semen data analyzed by traditional methods and a computer assisted semen analyzer and with hormone levels, were as follows. 1) In 21(7.2%) out of the 290 men, IgG-immunobead (IB) binding was observed in more than 50% of their motile spermatozoa; in 10(3.4%) of the 290, IgA-IB binding. 2) In all of the cases with proven fertility, the IB binding rate was less than 60% for IgG-IB, and less than 40% for IgA-IB. This suggested that antibodies attached to sperm could be among the factors in male infertility. 3) Antibodies attached to the sperm head with binding rate of less than 40% were not considered to be a causative factor in male infertility. 4) The incidence of positive IgG-IB was higher, and also in each case the IgG-IB binding rate was higher than the IgA-IB. 5) IgG-IB binding parts of each spermatozoon included IgA-IB binding parts. 6) No significant relationship was observed between the results of traditional semen analysis, LH.FSH.PRL levels or PENETRAK and the rates of IB binding.

Clinical course of early fetal loss and its chromosomal characteristics.

The relationship between types of chromosomal abnormalities and fetal development documented by ultrasonography was discussed in early spontaneous abortions. The subjects were 113 patients who had vaginal ultrasonography at least twice between 6 and 8 weeks of pregnancy, among 167 abortuses with chromosome analyzed. The results were also compared against those for 303 normally developing fetuses. The results obtained in the present study suggested that each fetus with a chromosomal abnormality succumbed at or after a specific stage of fetal development and that the fetal death might be the results of severe fetal growth suppression.

Cloning and characterization of the pectate lyase III gene of Erwinia carotovora Er.

A pectate lyase gene III (pel III) of Erwinia carotovora Er was cloned. The gene was expressed independently of a vector promoter in both E. carotovora Er and Escherichia coli. The pel III product was largely excreted in the culture medium of E. carotovora Er, while the product was only exported to the periplasmic space and was not excreted in the medium of E. coli. Nucleotide sequence analysis of pel III disclosed an open reading frame of 1,122 bp encoding a protein of 374 amino acids. The deduced amino acid sequence contained the N-terminal 30 amino acid sequence from the purified pectate lyase III (PL III) indicating the presence of a 22-amino-acid signal peptide. A putative ribosome-binding site was found to be 9 bp upstream of the start codon. The location of pel III was about 5.6 kb downstream of pel I. The PL III showed 80% homology in the amino acid sequence with the PL I of E. carotovora Er.

Effect of bifemelane on the intracellular pH and energy state of the ischemic brain.

4-(o-Benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane, CAS 90293-01-9, Celeport) has been reported to exert a protective effect on the brain against ischemic insults. However, the underlying mechanism of this action has not yet been fully elucidated. The effects of bifemelane on the intracellular pH (pHi) and energy metabolism of the ischemic brain were examined in Mongolian gerbils using in vivo 31P nuclear magnetic resonance spectroscopy. Transient global ischemia was produced by clipping both common carotid arteries for 45 min, and the brain was reperfused by releasing the clips. Bifemelane (10 or 20 mg/kg) or normal saline was administered intraperitoneally 30 min prior to the ischemia. During the ischemia, adenosine triphosphate (ATP) and phosphocreatine (PCr) were markedly reduced in association with an increase in inorganic phosphate (Pi) and decrease in pHi in both the control and bifemelane groups. The extents of energy disturbance and intracellular acidosis in the three groups were identical. After reperfusion, ATP, PCr, Pi and pHi recovered towards the pre-ischemic levels in all the groups. In the bifemelane groups, the recovery of pHi was significantly faster than in the control group. Of the two bifemelane groups, the 20 mg/kg group showed more excellent pHi recovery as compared to the 10 mg/kg group. The energy recovery in the three groups were almost identical, although the 20 mg/kg group showed some tendency towards faster recovery as compared to the control group. The present results suggest that bifemelane may accelerate recovery of the pHi after cerebral ischemia. Such an action may contribute to the cerebral protective effects of this drug against ischemic insults.