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.

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.

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.

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.

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.

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.

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.

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.

Study of experimental cervical spondylotic myelopathy.

UNLABELLED: Most cervical spondylotic myelopathy cases show insidious onset. Experimental cervical spondylotic myelopathy models were made using cats to analyze advancing patterns of myelopathy in the spinal cord and to identify the most responsible factor. Spinal cord compression was applied anteriorly using one screw through the C5 vertebral body or three screws through the C4, C5, and C6 vertebral bodies. Every few weeks, the screws were turned approximately 1 mm at a time up to approximately 50% of the spinal canal. At more than 6 months after final compression, limb function and evoked spinal cord potentials were evaluated. Thereafter, histologic examinations were performed. THE RESULTS: the single compression group showed normal limb function, but the multiple compression group showed sluggish gait. Histologically, mild neuronal decrease in the gray matter was noticeable in the single compression group. Conversely, the multiple compression group showed severe changes in the gray matter and significant changes in the posterior and anterior columns. There was a correlation between the evoked spinal cord potentials and histologic changes. It was clear that multiple compressions to the spinal cord caused more severe deterioration functionally and electrophysiologically as well as histologically than a single compression.

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.

Intraoperative monitoring for tethered spinal cord syndrome.

Functions of the lower extremities and bowel and bladder must be monitored during releasing surgery for tethered spinal cord syndrome, because neural elements are embedded in lipoma or anomalous tissues. Evoked muscle action potentials of the external anal sphincter muscle, external urethral sphincter muscle, and lower extremity muscles can indicate promptly whether or not neural elements are involved before the surgeon releases or cuts the tissue for cord release. It is also important to monitor vesical pressure because of different types of innervation from the external urethral and anal sphincters, in spite of slow reaction times of vesical pressure elevation. Evoked muscle action potentials indicate only motor function; therefore, it is more appropriate to record evoked spinal cord potentials from the spinal cord above a lumbosacral operative field, because sensory function can also be monitored. Evoked muscle action potentials are an easy and extremely sensitive monitoring system; nevertheless, monitoring of vesical pressure and evoked spinal cord potentials should also be done to achieve optimum monitoring. For the past 5 years, 10 patients have undergone cord-release surgery with such a monitoring system, and the results indicate no exacerbating case.

Sporeamicin A, a new macrolide antibiotic. I. Taxonomy, fermentation, isolation and characterization.

Sporeamicin A, a novel antibiotic, was isolated from the culture filtrate of an actinomycete. The producing organism, strain L53-18, was taxonomically assigned as a species of the genus Saccaropolyspora. The antibiotic was extracted with chloroform and was then purified by crystallization. It was obtained as colorless prisms from ethanolic solutions. Sporeamicin A exhibited a strong UV absorption peak at 276 nm. The molecular formula of sporeamicin A was determined to be C37H63NO12.

Sporeamicin A, a new macrolide antibiotic. II. Structure determination.

Structure of a novel antibiotic, sporeamicin A (SRM-A), was determined by a combination of spectroscopic and X-ray crystallographic studies. SRM-A has a unique structure containing a 2,3-dihydro-3-oxofuran moiety as part of a 14-membered macrolide ring.

Sporeamicin A, a new macrolide antibiotic. III. Biological properties.

Sporeamicin A is a new erythromycin-type antibiotic isolated from a species of Saccharopolyspora. It was active in vitro against a wide variety of Gram-positive bacteria. In vitro studies indicated that the sporeamicin A was stable in the presence of human serum, although it was bound to serum proteins. Sporeamicin A was effective in the mouse protection test against Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae. Sporeamicin A attained higher plasma and tissue levels in the rat than did erythromycin stearate.

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.

[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.

[Image analysis of the inner ear with CT and MR imaging: pre-operative assessment for cochlear implant surgery].

Recent progress in magnetic resonance imaging (MRI) has made it possible to obtain detailed images of the inner ear by delineating the lymphatic fluid within the labyrinth. We analyzed CT scans and MR images in 70 ears manifesting profound deafness owing to inner ear lesions and compared their detective ability for inner ear lesions. The following results were obtained. 1) CT scan examination showed slight to extensive ossification of the labyrinth in six ears (9%), whereas MRI examination revealed low to absent signal intensity of the inner ear in nine ears (13%). Therefore, it was concluded that MRI is more sensitive in detecting abnormalities of the inner ear than CT scan. 2) MRI provided useful information as to whether the cochlear turn is filled with lymphatic fluid or obstructed. This point was one of the greatest advantages of MRI over CT scan. 3) Abnormal findings in either or both the CT scan and the MRI were detected in suppurative labyrinthitis occurring secondary to chronic otitis media, bacterial meningitis and in inner ear trauma. However, such abnormal findings were not detected in patients with idiopathic progressive sensorineural hearing loss, ototoxicity or sudden deafness. These findings should be taken into consideration in pre-operative assessment of cochlear implant candidates.