Transmembrane topography of plasma membrane constituents in mung bean (Vigna radiata L.) hypocotyl cells. I. Transmembrane distribution of phospholipids.

The transmembrane distribution of phospholipids (PLs) in the plasma membrane (PM) of mung bean (Vigna radiata L.) hypocotyl cells was investigated using annexin V-fluorescein isothiocyanate, porcine pancreas phospholipase A(2), and (31)P-nuclear magnetic resonance (NMR) spectroscopy. Phosphatidylserine was not located on the cell surface of mung bean protoplasts. However, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid were found to be almost symmetrically distributed across right-side-out PM vesicles obtained by aqueous two-phase partitioning by porcine pancreas phospholipase A(2) assay. (31)P-NMR assay showed that the amount of PLs is about equal in the outer and the inner leaflets of the right-side-out PM vesicles. These results suggest that the topography of PM PLs might not contribute to well-known asymmetrical properties of the outer and inner surfaces of higher plant PMs. It is also indicated that inside-out PM vesicles created by Brij 58-treatment do not retain the native PL topography on dithionate reduction of 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled PLs incorporated in the PM vesicles.

Modulation in the activity of purified tonoplast H+-ATPase by tonoplast glycolipids prepared from cultured rice (Oryza sativa L. var. Boro) cells.

Glycolipids, phospholipids, and neutral lipids were extracted from the tonoplast fraction of cultured rice cells (Oryza sativa L. var. Boro). Acyl steryl glucoside (ASG) and glucocerebroside (GlcCer) were also prepared from this fraction. We determined the effects of these tonoplast lipids on the activity of H+-ATPase which was delipidated and purified from the tonoplast fraction. Exogenously added tonoplast phospholipids stimulated the activity of purified tonoplast H+-ATPase, but tonoplast glycolipids did not. When tonoplast glycolipids or tonoplast ASG was added in the presence of tonoplast phospholipids, they decreased the phospholipid-induced activation of the tonoplast H+-ATPase; tonoplast GlcCer only caused a small decrease. Steryl glucoside (SG) did not cause any decrease in this activation. Phospholipids, ASG, and GlcCer made up 35 mol%, 20 mol% and 7 mol% of the total lipids of the tonoplast fraction of cultured rice cells, respectively, and these glycolipid levels were enough to depress the phospholipid-induced activation of the tonoplast H+-ATPASE: These results revealed that H+-ATPase activity in the tonoplast may be modulated toward activation and depression by tonoplast phospholipids and glycolipids, respectively. The acylation of SG would be responsible for the depression in the phospholipid-induced H+-ATPase activity.

Endogenous 5-HT tonically inhibits spontaneous firing activity of dorsal hippocampus CA1 pyramidal neurons through stimulation of 5-HT(1A) receptors in quiet awake rats: in vivo electrophysiological evidence.

The present study was performed to examine an overall effect of endogenous serotonin (5-HT) on the spontaneous firing activity of the dorsal hippocampus CA1 pyramidal neurons in quiet awake rats. A selective 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xanecarboxamide (WAY-100635: 0.03-0.2 mg/kg, s.c.) significantly increased the firing activity. A depletion of 5-HT with parachlorophenylalanine (PCPA: 500 mg/kg/day x 3 days) completely abolished this increasing effect of WAY-100635. The baseline spike frequency of the PCPA-treated rats (3.90 +/- 0.39 Hz) was significantly higher than that of the vehicle-treated rats (2.09 +/- 0.19 Hz). A 5-HT(2A) antagonist ritanserin (1 mg/kg, i.p.) and a 5-HT(3/4) antagonist 2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester (SDZ-205557: 3 mg/kg, s.c.) did not modify the firing activity and the increasing effect of WAY-100635. These results suggest that, in quiet awake rats, endogenous 5-HT would tonically inhibit the spontaneous firing activity of the CA1 pyramidal neurons mainly through stimulating 5-HT(1A) receptors.

Mechanism of the chilling-induced decrease in proton pumping across the tonoplast of rice cells.

The ATP-generated proton pumping across tonoplast vesicles from chilling-sensitive Boro rice (Oryza sativa L. var. Boro) cultured cells was markedly decreased by chilling at 5 degrees C for 3 d. The membrane fluidity of core hydrophobic and surface hydrophilic regions of the lipid bilayer was measured by steady-state fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium 1,6-diphenyl-1,3,5-hexatriene and by electron spin resonance spectroscopy of 16- and 5-doxyl stearic acid, respectively. The fluidity of the surface region of the lipid bilayer of the tonoplast vesicles decreased by chilling. The fluidity of the surface region of the liposomes and the proton pumping across the reconstituted proteoliposomes with tonoplast H+-ATPase decreased with increasing content of the glycolipids. The proton pumping across chimera proteoliposomes was reduced by chilling only when it was reconstituted in the presence of tonoplast glycolipids from chilled Boro cells. These data suggest that the reduction in ATP-generated proton pumping across the tonoplast by chilling is due to the decrease in the fluidity of the surface region of the lipid bilayer of the tonoplast, which is caused by the changes in glycolipids.

Identification of the gene structure and promoter region of H+-translocating inorganic pyrophosphatase in rice (Oryza sativa L. ).

In order to determine the gene structure and promoter region of vacuolar H+-translocating inorganic pyrophosphatase (V-PPase), we isolated the genomic clones using a rice BAC library and probes derived from rice V-PPase cDNA (OVP1). The entire OVP1 gene is approx. 5.4 kb in length, and seven introns interrupt the coding sequence of OVP1. The first intron is extremely large (1869 bp), while the other introns are between 82 and 170 bp. A transcription initiation site, identified by a primer extension analysis, indicated the first exon to be 366 bp. A 1.1 kb fragment containing the 5'-flanking region of the first exon with the GUS reporter gene showed specific promoter activity in rice cells. These data show that the OVP1 gene is composed of eight exons and seven introns, and regulatory elements are present within 1.1 kb upstream from the first exon.

Anxiolytic 5-hydroxytryptamine1A agonists suppress firing activity of dorsal hippocampus CA1 pyramidal neurons through a postsynaptic mechanism: single-unit study in unanesthetized, unrestrained rats.

Recent behavioral studies indicate that conditioned fear response to contextual stimuli is reduced effectively by anxiolytic 5-hydroxytryptame (5-HT)1A agonists. Since the hippocampus seems to play an essential role in associative fear memories evoked by context, it is important to assess the effect of 5-HT1A agonists on pyramidal cell activity in the hippocampus. We examined the effects of 5-HT1A agonists on the spontaneous firing rate of hippocampal CA1 pyramidal neurons in unanesthetized, unrestrained rats. Systemic administration of selective 5-HT1A agonists, 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, ipsapirone, and flesinoxan produced a dose-dependent inhibition of neuronal activity. Putative 5-HT1A antagonists NAN-190 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine and (-)-pindolol did not change neuronal activity of CA1 pyramidal neurons. The suppression of neuronal activity by buspirone was antagonized by NAN-190 but not by (-)-pindolol. Lack of antagonistic activity of (-)-pindolol for the suppression of pyramidal neurons via a postsynaptic mechanism is consistent with the results of recent electrophysiological experiments in anesthetized rats. Pretreatment with parachlorphenylalanine did not change the spontaneous firing rates of hippocampal CA1 pyramidal neurons or abolish the suppressant effects of buspirone on these neurons. Taken together, the present results strongly suggest that suppression of the hippocampal CA1 pyramidal neuronal activity by anxiolytic 5-HT1A agonists in awake rats is mediated by postsynaptic 5-HT1A receptors located on pyramidal neurons.

Antiepileptic effects of allopurinol on EL mice are associated with changes in SOD isoenzyme activities.

We have investigated the potential antiepileptic action of superoxide dismutase (SOD) activities in the brain of the epileptic mutant EL mouse. EL mice which experienced frequent seizures (EL[s]) had abnormally low levels of SOD isoenzyme activity in the hippocampal area. Once epileptogenicity was established in these animals, activity of cyanide-sensitive Cu,Zn-SOD was maintained at significantly lower levels than in control mice. However, cyanide-insensitive Mn-SOD activity was not different from non-epileptic controls. In EL mice which had not experienced seizure provoking stimulations and exhibited no seizures (EL[ns]) there was moderately lower levels of SOD isoenzyme activities compared to controls. In spite of the low level of Cu,Zn-SOD activity in EL[s] mice, the Cu,Zn-SOD protein content was high in the hippocampus of these animals, suggesting that inactive Cu,Zn-SOD might be induced during development. After allopurinol (ALP) was given orally to EL[s] mice, Cu,Zn-SOD activities increased dramatically in the hippocampus and seizure activity was decreased. Even after 48 h, when antiepileptic action of ALP was lost, the SOD activity was maintained at the high level associated with initial ALP administration. EL[s] mice also showed DNA fragmentation in the hippocampal CA1 region and the parietal cortex, detected with in situ terminal transferase-mediated dUTP nick labeling with the aid of alkaliphosphatase or peroxidase. The degree of DNA fragmentation was less severe in EL[ns] mice. We propose that abnormalities in region specific Cu,Zn-SOD isoenzyme activity might produce free radicals, leading to DNA fragmentations and cell loss. This might contribute to hippocampal epileptogenesis in EL mice.

Genomic heterogeneity maps to tandem repeat sequences in the herpes simplex virus type 2 UL region.

Two Bam HI Y and W fragments in the unique long sequence (UL) of the herpes simplex virus type 2 (HSV-2) were found to be heterogeneous in size among clones derived from a single strain as well as from epidemiologically unrelated isolates. More detailed restriction maps of these BamHI fragments were constructed and the heterogeneous subfragments were defined, cloned, and sequenced in order to investigate the mechanism causing the size difference. The subfragment of BamHI Y contained a tandem repeat sequence consisting of different numbers of 15 bp, 5'AGGGGCGGCTGGGGC3' as one unit among three isolates, and the subfragment of BamHI W contained the other tandem repeat sequence, 9 bp, 5'CCTCCCGCC3'. In the UL of the HSV-2 strain, these tandem repeat sequences were conserved and each repeat number appeared to be highly variable through viral genome replication. These results showed that the fragment length polymorphisms in these regions were attributable to the variation of unit numbers of the tandem repeat sequences.

Effect of long-term administration of duloxetine on the function of serotonin and noradrenaline terminals in the rat brain.

Duloxetine, an inhibitor of both 5-hydroxytryptamine (5-HT) and noradrenaline (NA) reuptake processes, has been developed as a potential antidepressant drug. The present study was initiated to investigate the functioning of multiple components of the 5-HT and NA systems following the long-term administration of duloxetine. In rats treated for 21 days with duloxetine (20 mg/kg/day), the recovery times of dorsal hippocampus CA3 pyramidal neurons from microiontophoretic applications of 5-HT and NA were significantly increased, indicating ongoing reuptake blockade with the minipump in place delivering the drug. The remaining experiments were performed following a 48-h washout. Electrically evoked release of [3H]5-HT from preloaded slices was enhanced in the midbrain, presumably due to a desensitization of the somatodendritic 5-HT1D and 5-HT1A autoreceptors. In addition, evoked release of [3H]5-HT was increased in the hippocampus, which could have been due to the desensitization of the alpha2-adrenergic heteroreceptors located on the 5-HT terminals. In contrast, there was no change in the evoked release of [3H]5-HT in the frontal cortex despite decreased functioning of the 5-HT transporter found in this brain region. Similar to changes in 5-HT release, electrically evoked release of [3H]NA was enhanced in the hippocampus and frontal cortex of rats treated chronically with duloxetine. These increases in [3H]NA release were most likely due to the desensitization of the alpha2-adrenergic autoreceptor in the hippocampus and to the desensitization of the NA transporter in the frontal cortex, respectively. These data suggest that long-term administration of duloxetine is able to induce changes in the 5-HT and NA systems that lead to enhanced release of both 5-HT and NA in some limbic brain areas. Duloxetine, therefore, may be a useful antidepressant compound.

Preparation and characterization of an Actinomyces naeslundii aggregation factor that mediates coaggregation with Porphyromonas gingivalis.

Intergeneric coaggregation is responsible for the complexity of the microbiota in human dental plaque and is believed to be important in the initial bacterial colonization of the human oral cavity. Actinomyces naeslundii, an early colonizer of the tooth surface, may enhance subsequent colonization by Porphyromonas gingivalis which is associated with adult periodontitis. The purpose of this study was to isolate and characterize the A. naeslundii aggregation factor (AnAF) that mediates coaggregation with P. gingivalis. AnAF was isolated from A. naeslundii sonic extract (SE) by gel filtration on a Sephacryl S-400HR, by hydrophobic interaction chromatography on a HiTrap Octyl Sepharose 4FF, and by ion exchange chromatography on a HiTrap Q. The specific activity increased 12-fold with a yield of 2.5%. SDS-PAGE analysis of AnAF revealed a protein band of high molecular weight in excess of 200 kDa. Carbohydrate was detected as the only material coinciding with the protein band, indicating that the AnAF was a glycoprotein. Immunoblotting analysis indicated that AnAF directly bound to P. gingivalis cells. AnAF was sensitive to sodium metaperiodate treatment but not to heat or protease treatments. These results suggest that the AnAF carbohydrate component mediated coaggregation with P. gingivalis cells. AnAF also inhibited coaggregation with other periodontal disease-associated bacteria such as Prevotella intermedia, Fusobacterium nucleatum, Capnocytophaga ochracea, but not streptococci.

Purification, characterization, and cDNA cloning of a novel alpha-galactosidase from Mortierella vinacea.

A novel alpha-galactosidase, designated alpha-galactosidase II, was isolated from the culture filtrate of Mortierella vinacea. The molecular size of the purified enzyme estimated by gel filtration was 60 kDa, which agreed with that, 51-62 kDa, estimated by SDS-PAGE. The enzyme was thermolabile at neutral pH, but the addition of BSA to the enzyme solution at the concentration of 0.01% increased its stability considerably. The enzyme appears to be novel because it showed a distinct substrate specificity from other microbial alpha-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan, that is, the enzyme liberated not only side-chain alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotetraose but also terminal alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotriose. In addition, the enzyme acted on galactomannans effectively. alpha-Galactosidase II cDNA was cloned and its nucleotides sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 376 amino acid residues with a molecular mass of 41,334 Da. The derived amino acid sequence of the enzyme showed 31-49% sequence similarity with those of alpha-galactosidases from other origins.

Rapid reconstitution of tonoplast H(+)-translocating pyrophosphatase from cultured rice cells into liposomes.

We report the rapid and functional reconstitution of H(+)-pyrophosphatase (H(+)-PPase) from the tonoplast of cultured rice (Oryza sativa L.) cells to proteoliposomes. The CHAPS-solubilized H(+)-PPase was incorporated into liposomes by gel-filtration. Both the activities of PPi-hydrolysis and H(+)-pumping were influenced by the lipid-protein ratio and cholesterol.

Isolation and characterization of cDNAs encoding vacuolar H(+)-pyrophosphatase isoforms from rice (Oryza sativa L.).

The vacuolar H(+)-pyrophosphatase (V-PPase) is an electrogenic H+ pump, which was found in the plant vacuolar membrane. Two cDNA clones (OVP1 and OVP2) encoding the V-PPase were isolated from cultured rice (Oryza sativa L.) cells and subsequently sequenced. The sequence analysis has revealed that OVP1 contains 2316 nucleotides of open reading frame (ORF) and 362 nucleotides of the 3'-untranslated region, whereas OVP2 comprises 2304 nucleotides of ORF and 312 nucleotides of the 3'-untranslated region. The nucleotide sequences of ORF of OVP1 and OVP2 are 80.7% identical, and their 5'- and 3'-untranslated regions have 39.4% and 48.4% identity, respectively. The polypeptides encoded by the ORF of OVP1 and OVP2 contain 771 and 767 amino acids, respectively, and the sequences of the OVP proteins are very similar to those of other V-PPases, which are shown to have 85-91% homology. Chromosomal mapping by RFLP techniques demonstrates that OVP1 and OVP2 are isoforms encoded by different genes. Both OVP1 and OVP2 are mapped on the same chromosome (chromosome 6) to a distance of ca.90 cM. Northern analysis indicates that the OVP1 and OVP2 are also expressed in intact rice plants and OVP2 shows higher expression in the calli than the roots and shoots, compared to OVP1. These results show that at least two genes encoding the V-PPases are present in rice genome and their expressions are probably regulated in a different manner.

Blockade of the serotonin and norepinephrine uptake processes by duloxetine: in vitro and in vivo studies in the rat brain.

In in vitro uptake experiments, duloxetine inhibited [3H]5-hydroxytryptamine (5-HT) and [3H]norepinephrine (NE) uptake in hippocampus slices of control rats with IC50 values of 28 and 46 nM, respectively. The uptake of both[3H]5-HT and [3H]NE was equipotently inhibited in hippocampus slices prepared from rats treated for 2 days with different doses of duloxetine (5, 10, 15 and 20 mg/kg/day s.c.). In in vivo electrophysiological experiments in the hippocampus, the effects of duloxetine on the suppression of CA3 pyramidal neuronal firing activity by microiontophoretically applied 5-HT and NE were examined with two modes of administration. Five successive i.v. injections (2 mg/kg each) significantly and dose-dependently prolonged the recovery time of the firing activity of hippocampus CA3 pyramidal neurons from the 5-HT applications. A 2-day treatment (10, 15 and 20 mg/kg/day s.c.) also increased the recovery time in a dose-dependent manner. Whereas the recovery time from NE applications was unaffected by low doses of duloxetine (2 mg/kg i.v.; 10 mg/kg/day for 2 days), it was prolonged significantly by higher doses (8 and 1 0 mg/kg iv.; 20 mg/kg/day for 2 days). Acute i.v. injections of duloxetine suppressed the spontaneous firing activity of dorsal raphe 5-HT and locus ceruleus NE neurons with ED50 values of 99 and 475 microgram/kg, respectively. Taken together, the present results confirmed that duloxetine is a dual 5-HT/NE uptake inhibitor. Furthermore, the results obtained in in vivo experiments indicate that duloxetine has a preferential inhibitory effect on the 5-HT transporter.

Purification and some properties of alpha-galactosidase from Penicillium purpurogenum.

alpha-Galactosidase was purified by ion-exchange chromatographies on DEAE-cellulose and SE-cellulose columns from the culture filtrate of Penicillium purpurogenum No. 618. The final preparation was judged homogeneous by SDS-PAGE and its molecular mass and isoelectric point were estimated to be 67 kDa and 4.1, respectively. The N-terminal amino acid sequence of the enzyme was analyzed and aligned with those of other alpha-galactosidases. In addition, the enzyme acted on the stubbed alpha-galactosyl residue connected to the beta-1,4-manno-oligosaccharide chain, indicating that this specificity was quite different from that of Mortierella vinacea alpha-galactosidase.

Nucleotide sequence of alpha-galactosidase cDNA from Mortierella vinacea.

To analyze the primary structure of Mortierella vinacea alpha-galactosidase, a cDNA library of M. vinacea mRNA in lambda gt10 was constructed. A clone, which has an insert size of about 1.4 kilobase pairs, was found to contain the coding region of the mature enzyme. The deduced amino acid sequence share that the mature enzyme consisted of 397 amino acid residues with a molecular mass of 44,350 Da. The sequence identity of the mature enzyme with alpha-galactosidases from Saccharomyces carlsbergensis, Cyamopsis tetragonoloba (guar), and human were 47%, 43%, and 34%, respectively.

Identification and characterization of a family of genes for the plasma membrane H(+)-ATPase of Oryza sativa L.

A cDNA clone (cOSA2) encoding a plasma membrane H(+)-ATPase was isolated from rice. Southern blot analysis indicated that the genes that corresponds to cOSA2 was different from that to cOSA1. Northern blots revealed OSA2 mRNA in roots, calli and shoots. OSA1 transcripts were detected only by RT-PCR in these tissues.

Effects of several 5-HT1A agonists on hippocampal rhythmical slow activity in unanesthetized rats.

We examined the effect of 5-hydroxytryptamine (5-HT)1A agonists on walking related, atropine-resistant, rhythmical slow activity (wr-RSA) of the hippocampus in rats. Selective 5-HT1A agonists, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), flesinoxan, buspirone and ipsapirone significantly decreased the power value of 7-9 Hz band activity and the median frequency of wr-RSA. The order of potency was 8-OH-DPAT > flesinoxan = buspirone in power reduction. The 5-HT1A antagonists, (-)pindolol, (-)propranolol and spiperone, inhibited the effect of 8-OH-DPAT on wr-RSA. Pretreatment with parachlorophenylalanine did not abolish the effect of 8-OH-DPAT. These results indicate that 5-HT1A agonists reduce both power and median frequency values of wr-RSA through activation of post-synaptic 5-HT1A receptors in the forebrain in unanesthetized rats, in vivo.

Effects of zopiclone, triazolam, and nitrazepam on standing steadiness.

We examined the effects of the nonbenzodiazepine hypnotic zopiclone and the benzodiazepine hypnotics triazolam and nitrazepam on standing steadiness. Eight healthy volunteers received placebo, zopiclone (7.5 mg), triazolam (0.25 mg), and nitrazepam (5 mg) in a random-order, double-blind crossover design. Postural sway was assessed before and 1 and 2 h after drug administration using a stabilometer connected to a microcomputer. Triazolam significantly impaired standing steadiness. Zopiclone also impaired standing steadiness but the degree of impairment seemed to be less marked. Nitrazepam 5 mg had no significant effects on postural sway. Triazolam 0.25 mg, zopiclone 7.5 mg, and nitrazepam 5 mg, which are reported to be equipotent to each other as hypnotics, are not equipotent with respect to their effects on postural sway.