Heat-killed Lactobacillus helveticus improves mood states: a randomised, double-blind, placebo-controlled study.

We investigated the effects of heat-killed Lactobacillus helveticus MCC1848 on daily mood states in healthy young adults. Participants (n=58) were randomised to receive heat-killed L. helveticus MCC1848 powder or placebo powder for 4 weeks. During the study period, adverse events were recorded in the participant diary. Mood states were assessed before and 2 and 4 weeks after initiation of the intervention. The primary outcomes were the shortened version of the Profile of Mood States 2 (POMS 2) scores. Secondary outcomes included other mood state (State-Trait Anxiety Inventory (STAI); visual analogue scale (VAS)), quality of life (acute form of the SF-36v2), sleep (Athens Insomnia Scale (AIS)) and fatigue (Chalder Fatigue Scale (CFS)) scores. Four weeks of heat-killed L. helveticus MCC1848 intake, compared to placebo, significantly improved the shortened version of the POMS 2 'friendliness' and the VAS 'relaxed' scores, which are two indicators of positive mood states. On the other hand, heat-killed L. helveticus MCC1848 intake had no significant effects on negative mood state items (e.g. anger, nervousness, confusion) assessed by the shortened version of the POMS 2, STAI and VAS. AIS and CFS scores also showed no significant differences. No adverse effects were observed with 4 weeks of heat-killed L. helveticus MCC1848 intake. These results suggest that daily consumption of heat-killed L. helveticus MCC1848 is safe and has the potential to improve positive mood states. UMIN Clinical Trial Registry: UMIN000043697.

Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease.

Hirschsprung disease (HSCR) is sometimes associated with a set of characteristics including mental retardation, microcephaly, and distinct facial features, but the gene mutated in this condition has not yet been identified. Here we report that mutations in SIP1, encoding Smad interacting protein-1, cause disease in a series of cases. SIP1 is located in the deleted segment at 2q22 from a patient with a de novo t(2;13)(q22;q22) translocation. SIP1 seems to have crucial roles in normal embryonic neural and neural crest development.

Cloning and characterization of the ddc homolog encoding L-2,4-diaminobutyrate decarboxylase in Enterobacter aerogenes.

L-2,4-diaminobutyrate decarboxylase (DABA DC) catalyzes the formation of 1,3-diaminopropane (DAP) from DABA. In the present study, the ddc gene encoding DABA DC from Enterobacter aerogenes ATCC 13048 was cloned and characterized. Determination of the nucleotide sequence revealed an open reading frame of 1470 bp encoding a 53659-Da protein of 490 amino acids, whose deduced NH2-terminal sequence was identical to that of purified DABA DC from E. aerogenes. The deduced amino acid sequence was highly similar to those of Acinetobacter baumannii and Haemophilus influenzae DABA DCs encoded by the ddc genes. The lysine-307 of the E. aerogenes DABA DC was identified as the pyridoxal 5'-phosphate binding residue by site-directed mutagenesis. Furthermore, PCR analysis revealed the distribution of E. aerogenes ddc homologs in some other species of Enterobacteriaceae. Such a relatively wide occurrence of the ddc homologs implies biological significance of DABA DC and its product DAP.

Role of Atf1 and Pap1 in the induction of the catalase gene of fission yeast schizosaccharomyces pombe.

We examined the induction of the catalase gene (ctt1(+)) of fission yeast Schizosaccharomyces pombe in response to several stresses by using mutants of transcription factors (Atf1 and Pap1) and a series of deletion mutants of the ctt1(+) promoter region. A transcription factor, Atf1, and its binding site are necessary for the induction of ctt1(+) by osmotic stress, UV irradiation, and heat shock. Induction by menadione treatment, which produces superoxide anion, required element A, the region from -111 to -90 (numbered with the transcription start site as +1). The factor responsible for the induction of the gene by oxidative stress via element A was identified as the transcription factor Pap1. We also found that Atf1 is activated by menadione treatment in pap1 mutant cells, although it is not activated by menadione treatment in pap1(+) cells. The activity of catalase is not increased in pap1 cells by several stresses, despite mRNA induction, suggesting that Pap1 plays some role in the expression of catalase activity.

Schizosaccharomyces pombe homologue of glutathione peroxidase, which does not contain selenocysteine, is induced by several stresses and works as an antioxidant.

We have cloned a gene of Schizosaccharomyces pombe homologues to the glutathione peroxidase gene. The cloned gene, named gpx1(+), encoded a protein that was 158 amino acids in length and had a molecular mass of 18 kDa. The gpx1(+) gene is homologous with many glutathione peroxidase genes but the selenocysteine codon (UGA) position of mammalian genes is a cysteine codon (UGU) in S. pombe. gpx1(+) mRNA was induced by various stresses, including oxidative stress, osmostress and heat stress. These stresses activate the Wis1-Sty1/Spc1 MAP kinase cascade in S. pombe. Transcriptional factors Atf1 and Pap1 are under the control of this MAP kinase. In the disruption of the atf1(+) gene, gpx1(+) was not transcribed or induced. However, the expression of gpx1(+) was not affected by the disruption of the pap1(+) gene. These results indicated that gpx1(+) was under the control of transcription factor Atf1. Catalase can detoxicate H(2)O(2) in the same way as GPx and the disruptant of the catalase gene of S. pombe is hypersensitive to H(2)O(2). The catalase gene disruptant of S. pombe harbouring multicopy plasmid containing gpx1(+) restored the hypersensitivity to H(2)O(2) of the catalase gene disruptant. These results suggest that Gpx1 acts as a scavenger of H(2)O(2) in vivo.

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.

Identification of the catalase gene promoter region involved in superinduction in Schizosaccharomyces pombe caused by cycloheximide and hydrogen peroxide.

Superinduction of the catalase gene was observed in Schizosaccharomyces pombe cells treated with cycloheximide and hydrogen peroxide. The promoter analysis of the catalase gene revealed that element A (the region from -111 to -90, numbered with the transcription start site as +1), involved in the induction of the gene under oxidative stress, was required for superinduction by hydrogen peroxide and cycloheximide. Although Atf1 is a transcription factor responsible for the induction of the catalase gene by several stresses, a disruptant of atf1 exhibited superinduction. Moreover, in a deletion mutant that lacks element A but has an Atf1 binding site, the cells treated with hydrogen peroxide and cycloheximide expressed as much catalase mRNA as those treated with hydrogen peroxide alone. This suggests that cycloheximide does not stabilize the catalase mRNA but enhances the transcription via element A. Staurosporine, a strong inhibitor of protein phosphorylation, did not inhibit superinduction.

Two distinct upstream regions are involved in expression of the catalase gene in Schizosaccharomyces pombe in response to oxidative stress.

The DNA region responsible for the induction of the catalase gene of Schizosaccharomyces pombe in response to oxidative stress was determined by constructing a series of deletions in the 5'-flanking region of the gene. Cells having deletion -672 (numbered with the transcription start site as +1) to -111 showed no significant difference in catalase expression from the wild-type cells. Cells having deletion -672 to -89 showed reduced basal expression of the catalase mRNA, but retained the ability of induction in response to oxidative stress. Cells having deletion -672 to -55 completely lost the ability to express the catalase mRNA. These results suggested that two regions, -89 to -55 and -111 to -89, are involved in expression of the catalase gene. The DNA region of -89 to -55 overlapped with the Atf1 binding sequence. The Atf1 is a bZIP transcription factor with an important role in stress response under the control of the Spc1 mitogen activated protein (MAP) kinase. Introduction of the atf1(-) or spc1(-) mutation into the mutant having a deletion in -672 to -89 completely abolished the expression of the catalase mRNA. This result indicated that the Spc1-Atf1 cascade is involved in expression of the catalase gene through the region of -89 to -55. In mutants spc1(-) and atf1(-), basal expression and induction by hydrogen peroxide of catalase mRNA were observed. These results revealed that not only the Atf1 binding site but also another DNA element independent of the Spc1-Atf1 pathway is involved in the expression of the catalase gene in response to oxidative stress in S. pombe. Proteins that bound specifically to each DNA element existed in the cell extract of the wild-type S. pombe.

Occurrence of a novel L-2,4-diaminobutyrate decarboxylase activity in some species of Enterobacteriaceae, and purification and characterization of the enzymes of Enterobacter aerogenes and Serratia marcescens.

L-2,4-Diaminobutyrate decarboxylase (DABA DC) is a novel enzyme yielding 1,3-diaminopropane (DAP) from DABA, which has previously been purified from strains of the genera Vibrio and Acinetobacter. In this study, we also detected DABA DC activity in the species of Enterobacteriaceae: E. aerogenes, E. cloacae, E. agglomerans, Serratia marcescens, S. liquefaciens, Klebsiella pneumoniace, K. oxytoca and Citrobacter freundii, all of which produced DAP in sufficient amounts. Subsequently, the DABA DCs of E. aerogenes and S. marcescens were purified to homogeneity and characterized. Two separate enzymes had similar properties with respect to chromatographic behaviors, and were a dimer with subunits of identical molecular mass of about 51 kDa. The maximal activity of each enzyme was obtained at pH 8.0-8.25. Both enzymes required pyridoxal 5'-phosphate and Mg2+ for full activity, and were highly specific for L-DABA. There was immunological similarity, but not identity between these proteins, as determined by Ouchterlony double diffusion analysis with antiserum against the E. aerogenes DABA DC. They showed the same N-terminal amino acid sequence up to the 8th residue (S-K-L-N-P-I-L-A-). These enzymes were different in molecular mass, N-terminal amino acid sequence and antigenicity from DABA DCs of Acientobacter and Vibrio species.

Monitoring of anterior cervical spinal cord function.

Anterior surgery is frequently chosen for treatment of cervical myelopathy. However, intraoperative spinal cord recording has rarely been used to monitor the function of the ventral columns. We report a method of monitoring evoked spinal cord potentials useful for detection of minor injury of the anterior spinal cord. Evoked spinal cord potentials elicited in cats by thoracic spinal cord and labyrinth stimulation were studied. Evoked intraspinal field potentials recorded after labyrinth stimulation were confirmed to originate from the vestibulospinal tract in the ventral columns. Low-amplitude potentials were recorded from the posterior epidural space. However, this method has not been used clinically because of difficulty in obtaining selective stimulation in humans. Spinal cord potentials evoked by thoracic stimulation were recorded from the anterior and posterior epidural spaces. The amplitude of the potentials was large enough to permit quantitation of neural function. We confirmed that anterior recording was more sensitive in detecting ventral column injury than posterior recording was. Based on these findings, we used anterior recording from the disc clinically for anterior spinal cord monitoring during anterior cervical surgery.

The effect of Helicobacter pylori on gastric acid secretion by isolated parietal cells from a guinea pig. Association with production of vacuolating toxin by H. pylori.

BACKGROUND: One of the features of Helicobacter pylori infection in the human stomach seems to be disordered gastric acid secretion. The effect of vacuolating toxin (VT) produced by H. pylori on gastric acid secretion was examined. METHODS: VT(+)(toxigenic) and VT(-)(nontoxigenic) strains of H. pylori were cultured in brucella broth. The culture supernatant was added to isolated parietal cells, and acid secretion and intracellular adenosine 3'5'-cyclic phosphate (cAMP) and Ca2+ levels were measured with the 14C-aminopyrine (14C-AP) method, with 125I radioimmunoassay (RIA), and with the fura-2 fluorescence method, respectively. RESULTS: In the VT(+) strain a considerable inhibitory effect on 14C-AP accumulation was observed. However, the VT(-) strain had no significant effect on intracellular c-AMP and Ca2+. CONCLUSIONS: The VT(+) strain of H. pylori has an inhibitory effect on gastric acid secretion, whereas the VT(-) strain does not. This inhibitory effect was not associated with the response of second messengers. It is speculated that VT produced by H. pylori has a direct action on H(+)-K+ adenosine triphosphatase in parietal cells.

Roles of sigma receptors in isolated guinea pig parietal cells.

Sigma receptor involvement in the oxyntic mechanism of gastric parietal cells was investigated using isolated guinea pig parietal cells. Using the 14C-aminopyrine accumulation method, di(orthotolyl)guanidine (DTG), a sigma receptor agonist, demonstrated peak stimulation of acid production at a concentration of 5 x 10(-5) M. The DTG-induced stimulatory effect on 14C-aminopyrine accumulation in preparations enriched in parietal cells was inhibited by haloperidol, a DTG inhibitor, and omeprazole, a proton pump inhibitor, in a concentration-dependent manner. Assessment of the interaction between DTG and agonists for histamine, gastrin, and muscarine receptors demonstrated a potentiating interaction effect on acid production only when parietal cells were exposed to both DTG and histamine. A receptor-binding assay using 3H-DTG revealed two sigma receptor subtypes, sigma 1 and sigma 2, on the crude membranes of parietal cells. There was a direct relationship between DTG dose and intracellular Ca2+ concentration, but there was no change in intracellular cyclic adenosine 5'-monophosphate (cAMP) concentration when parietal cells were exposed to DTG. These results demonstrate that sigma receptors exist on guinea pig gastric parietal cells and suggest that they play a role in acid production via an increase in intracellular Ca2+ concentration.

Transcriptional regulation of catalase gene in the fission yeast Schizosaccharomyces pombe: molecular cloning of the catalase gene and northern blot analyses of the transcript.

Exposure of Schizosaccharomyces pombe cells to various stresses including 0.2 mM hydrogen peroxide, 50 microM menadione, 10 J/m2 of UV irradiation at 255 nm, and high osmolarity (0.5 M sorbitol or 0.3 M NaCl) induces catalase [EC 1.11.1.6] activity. A part of the catalase gene of S. pombe was amplified by PCR with oligonucleotide primers designed from amino acid sequences conserved in several species of catalases. The catalase gene including its flanking sequence of S. pombe was cloned from a genomic DNA library of S. pombe, which was constructed on the EMBL3 vector, using the PCR-amplified DNA as a radioactive probe. A 3.5 kb HindIII fragment, which hybridized with the PCR-amplified probe, was subcloned into pUC19 and sequenced. The fragment contains one long open reading frame without any intron. The polypeptide deduced from the nucleotide sequence consists of 512 amino acid residues and is homologous to several other catalases. Amino acid sequences of the proteolytic peptides obtained from the purified catalase of S. pombe coincided with the amino acid sequence predicted from the DNA sequence. Transcription of this gene starts at 370 bases upstream of the initiation methionine codon. Northern blot analyses of the catalase mRNA revealed that the stresses which induce the catalase activity also induce the transcription of the catalase gene. The induction of the catalase mRNA by hydrogen peroxide is not inhibited by cycloheximide or staurosporine.

Molecular cloning and nucleotide sequencing of the gamma-glutamylcysteine synthetase gene of the fission yeast Schizosaccharomyces pombe.

A DNA fragment encoding gamma-glutamylcysteine synthetase [EC 6.3.2.2] of Schizosaccharomyces pombe was cloned by complementation of the cadmium hypersensitivity of a S. pombe mutant deficient in the enzyme. Sequence analysis of the cloned DNA revealed that the enzyme was consisted of 669 amino acid residues and was homologous to the enzymes of human liver, rat kidney, and Saccharomyces cerevisiae. The deduced amino acid sequence coincides with the amino acid sequences of the proteolytic peptides obtained from the purified enzyme. A cysteine residue was deduced to be important for catalytic activity by comparing the amino acid sequences of the enzymes of the four species. The gene contains one intron and the splicing point was confirmed by sequencing a cDNA amplified by PCR. Northern blot analysis showed an RNA of 2,200 bases hybridized with the cloned gene.

Giant sacral cysts with neurogenic bladder.

Most sacral cysts are accidentally found on lumbar myelograms and are usually asymptomatic. We operated on two patients with giant sacral cysts from S3 nerve roots who complained of neurogenic bladder and perianal sensory disturbance as well as buttock pain. Morphology of these cysts and intraoperative electrophysiological findings of nerve conduction block showed two kinds of pathogenesis causing these neurological symptoms. One was attributed to conduction block of more caudal sacral nerves squeezed between these giant sacral cysts and the other was due to degeneration of nerve root fibers involved in the sacral cyst walls. Postoperatively, buttock pain and perianal hypesthesia were resolved, but the neurogenic bladder showed only partial recovery.

[Inhibitory mechanism of experimental stress ulcer in spontaneously hypertensive rats (SHR) from the view point of mucosal blood flow].

Stress ulcer formation is reportedly much less frequent in SHR than in normotensive control rats (Wistar Kyoto Rat: WKY). The purpose of this study was to investigate the mechanism of maintenance of gastric mucosal blood flow (GMBF) during imposed stress in SHR. In stressed-only SHR, GMBF did not significantly change during water immersion and restraint conditions and ulcer index (UI) was significantly lower than that of WKY. Stress conditions led to a fall in blood pressure and a gradual fall in heart rate in WKY and SHR. It was assumed that the changes in blood pressure and heart rate during stress were due to vagal hyperfunction. The catecholamine level in the fundic gland of the gastric tissue was higher in the non-stressed SHR than in the non-stressed WKY. The administration of 6-hydroxydopamine to SHR produced a significant reduction in GMBF during stress conditions and UI was significantly higher in this group than in the stressed-only SHR. In SHR treated with nifedipine, UI was lower than that of the control group and GMBF showed no significant change compared with the stressed-only SHR. However, the administration of verapamil produced a significant reduction in GMBF during stress conditions and increased UI. The norepinephrine and dopamine levels of the groups treated with verapamil were significantly lower than those in the groups treated with nifedipine. These results suggest that local regulation of gastric mucosa mediated by sympathetic hyperfunction in SHR is more important for the maintenance of GMBF during stress conditions than changes in peripheral artery resistance.

Neuroradiologic and electrophysiologic assessment of cervical spondylotic amyotrophy.

Dissociated motor loss due to cervical spondylosis and disc herniation was evaluated in 10 patients who presented with left deltoid paresis in the absence of sensory deficits or myelopathy. All of these cases underwent cervical anterior decompression. Based on magnetic resonance imaging, computed tomography myelography, and computed tomography discography, patients were divided into two pathologic types: The first showed focal bony spur and disc herniation with axial cord rotation and nerve root compression, and the second demonstrated ventral cord flattening. Electrophysiologic studies included evoked spinal potentials, motor evoked potentials, and evoked muscle action potentials. Motor evoked potentials, recorded epidurally from the ventral aspect of the thecal sac and the nerve root within the anterior discectomy or vertebrectomy sites, proved clinically most useful. Combining the latest available neuroradiologic and electrophysiologic information, 4 types of neural injury associated with deltoid pareses were identified in the 10 patients. The first included isolated C5 nerve root lesions; the second, C6 nerve root lesions; the third, both C5 and C6 nerve root lesions, and finally, intrinsic cord pathology.

Molecular cloning and nucleotide sequencing of Schizosaccharomyces pombe homologue of the class II fructose-1,6-bisphosphate aldolase gene.

DNA fragment containing Schizosaccharomyces pombe homologue of the class II fructose-1,6-bisphosphate aldolase gene was cloned and sequenced. A long open reading frame, which encodes a polypeptide of 358 amino acid residues, was found in the sequence. Amino acid sequence deduced from the nucleotide sequence is 63% homologous to the amino acid sequence of the enzyme of Saccharomyces cerevisiae. Northern blot analysis revealed that 1.3 kb poly(A)+ RNA is transcribed from this DNA sequence.

Fate of cadmium bound to phytochelatin in rats.

The fate cadmium(Cd) bound to phytochelatin [PC, (gamma-Glu-Cys)n-Gly)] was studied in rats using synthesized 109Cd-PC. Less Cd was absorbed through the digestive tracts than CdCl2, but the ratio of renal Cd to hepatic Cd was higher. After parenteral administration of Cd-PC, Cd was distributed mainly in the liver, kidney, small intestine and pancreas. More Cd was found in the kidney than the liver after Cd-PC (n = 5) administration. Most of the Cd was bound to the high molecular weight fraction in the hepatic cytosol 0.5 hr after administration and moved to the metallothionein fraction at 6 hr. The tissue distribution of Cd was not affected even when free PC (n = 5) was administered 3 hr after or before Cd injection. The distribution in the kidney increased only in the case of the simultaneous administration of Cd with PC. These findings show that the absorbance of Cd bound to PC from the alimentary tract is lower than that of CdCl2 although absorbed Cd is distributed to the kidney more than CdCl2, and Cd is liberated from PC soon after uptake by the cells.