Biomedical subjects
Publications and source records attributed to N Kamimura.
Preliminary screening of antiviral AIDS drugs has been carried out using three different in vitro assay systems. Among 246 samples of different origin tested, six were shown to inhibit the growth of HIV in vitro. Two of the positive samples have hopeful signs, as the ranges of effective doses are wider than those of most of positive samples which had been found by us.
The store-mediated Ca2+ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca2+ activated ionic membrane currents. In the cells where intracellular Ca2+ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca2(+)-free extracellular condition, an employment of external Ca2+ in the absence of ACh caused a sustained increase of the K+ current without affecting the Cl- current. A renewed ACh challenge without external Ca2+ caused repetitive spikes of both K+ and Cl- currents due to the Ca2+ release. SK & F 96365 inhibited the generation of the sustained K+ current and refilling of the Ca2+ store following the Ca2+ readmission. It is suggested that the Ca2+ enters the cell through the store-mediated pathway new the K+ channels and is taken up by the store. Thus, only Ca2+ released from the store can activate both the K+ and Cl- currents.
In order to examine whether Ca2+ entry is directly involved in controlling exocrine secretion, the Ca(2+)-activated Cl- currents were recorded in single and clusters of rat submandibular gland cells using the whole-cell patch-clamp method. Extracellularly applied acetylcholine (ACh, 10 nM) as well as intracellularly applied GTP gamma S and InsP3 caused repetitive transients of the Cl- currents activated by intracellular Ca2+. These responses occurred also in the absence of external Ca2+, but disappeared after several minutes. Readmission of Ca2+ to the extracellular solution restored the repetitive current transients, while introduction of Sr2+ failed to restore the current signals in spite of the presence of Sr2+ entry detected by microfluorimetry. On the other hand, direct application of Sr2+ to the cell inside caused activation of the Cl- currents although less effectively than Ca2+. When Ca2+ was introduced to the extracellular solution during an interruption of ACh stimulation after the ACh-induced depletion of intracellular Ca2+ store, the Cl- current was not elicited. However, a subsequent challenge with ACh at the same concentration in the absence of extracellular Ca2+ caused repetitive transient Cl- currents. The results suggest that in this cell type the stimulated Ca2+ entry does not by itself activate the Cl- currents but activates them indirectly by triggering Ca2+ release from the intracellular Ca2+ store which may take up Ca2+ soon after the Ca2+ entry.
The influence of boiling water treatment on the surface roughness and surface microstructure of set gypsums was investigated. Typical surfaces before and after immersion in boiling water were compared by means of SEM observation, the Knoop hardness test, and a surface roughness test. The surfaces of set gypsums were rougher than that of an acrylic resin plate, and after immersion in boiling water, highly roughened surfaces and thinner crystal bodies were observed on each specimen under SEM. The knoop hardness of set gypsums was considerably lowered after boiling water immersion. That of die stones was the same or lower than set dental stones. The results showed that even brief immersion in boiling water had profound effects on the dental stone cast, resulting in rougher surfaces and lower hardness of set gypsums.
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Saccharomyces cerevisiae synthesizes ergosterol via squalene, but squalene is hardly detected in aerobically grown cells. To obtain a stable squalene-accumulating yeast strain, we attempted to disrupt a gene required in the conversion of squalene to ergosterol, by homologous recombination with a short piece of the gene fragment conjugated with an integration plasmid vector carrying the LEU2 gene. Two mutants that required ergosterol at least for fast growth were isolated. In an aerobic cultivation and with ergosterol supplementation, the two mutants accumulated squalene up to 5 mg/g dry cells. Southern hybridization analysis indicated that both mutants had acquired the vector DNA integrated in the same gene, or nearby genes, on chromosome 12.
Preliminary screening for antiviral AIDS drugs has been carried out using three different in vitro assay systems. Among 154 samples tested, nine were found to inhibit the growth of HIV in vitro. One of nine has a hopeful sign, as the range of effective doses of the sample is wide.
A 75-year-old female was admitted to our hospital because of fever and hypotension. The peripheral blood showed 400 leukocytes/microliters with 13,000/microliters platelets. Bone marrow puncture revealed that NCC stood at 14,000 with 50.0% blasts. The surface characters of the blasts were CD13+, CD33+, and HLA-DR+, and blood culture tests were positive. Coagulation tests revealed DIC. Based on the foregoing results, hypoplastic leukemia was diagnosed accompanied by sepsis and DIC, and was placed on the concomitant administration of a combination of low dose Ara-C and M-CSF. After 14 days of Ara-C administration and 26 days of M-CSF, her clinical symptoms improved, with the peripheral blood showing a WBC of 2,800/microliters and platelet count of 111,000/microliters. The percentage of myeloblasts decreased to 7.0%. After the administration of Ara-C was suspended for 2 weeks, another course of low dose Ara-C plus M-CSF administration was carried out and the patient achieved full remission. M-CSF stimulates not only the production of monocytes but increases the number of neutrophils and platelets through monocytes. It is also expected that tumoricidal activity may be realized by the activation of monocytes. In this patients, the concomitant administration of M-CSF and low dose Ara-C was remarkably effective in treating hypoplastic leukemia with severe complication. This result suggests that M-CSF will be useful for the treatment of leukemia.
Preliminary screening for antiviral AIDS drugs was carried out using three different in vitro assay systems. Among 104 samples tested, six were found to inhibit the growth of HIV in vitro. Four of six were acidic polysaccharides of sea weed origin and one was a well-known anti HIV chemical whose anti-HIV activity has not been reported.
To determine whether mitochondrial DNA (mtDNA) fragments found within the nucleus are transcribed, we have differentially screened a HeLaTG cDNA library. A clone that hybridized to mtDNA as well as to c-myc was identified. Analysis of the cDNA disclosed that it contained a mtDNA sequence, encoding cytochrome-c oxidase subunit III (coxIII) that was contiguous with and 5' of a c-myc sequence corresponding to part of exon 2 and exon 3. Hybridization of ScaI-digested DNA with a 1.05 kb c-myc probe revealed a unique band in HeLaTG cells, as well as a band common to HeLaTG and 13 other cell types examined. Solution hybridization of HeLaTG RNA with a radiolabeled, single-stranded cDNA probe containing the coxIII-c-myc junction demonstrated a nuclease-resistant band that matched the full length of the junctional cDNA probe. A smaller band that equaled the size of the c-myc portion alone was also detected. Only the smaller band coinciding with the c-myc sequences was protected from nuclease digestion by RNA from other cells. When a radiolabeled probe synthesized in the opposite orientation was used, nuclease-resistant bands equal in length to the coxIII portion of the probe were detected after hybridization with RNA from all cells. These results indicate that insertion of mtDNA fragments into nuclear genes occurs and that subsequent transcription of a 'chimaeric' or 'fusion' mRNA containing both mitochondrial and nuclear sequences can ensue.
The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.
We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.
This study consisted of 1) molecular deletion analyses in patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) using the entire cDNA for the DMD gene as hybridization probes, 2) RFLP analyses in a large number of Japanese normal women using 11 DMD-linked cloned DNAs as probes, and 3) segregation analyses with these RFLP data in 17 DMD families in which prenatal or carrier diagnosis was required. The deletion study showed that 18 (43%) of 42 male DMD patients had a deletion within the DMD gene, while no detectable deletion was found in 3 BMD patients. These deletions were preferentially observed at the 5' end of the DMD gene, while no deletion was found in the 3' portion of the gene. Of a total of 15 RFLPs detected with the 11 probes, one was a new RFLP (probe/enzyme: P20/MspI). In 6 RFLPs, the allele frequencies in the Japanese were statistically different from those in the Caucasian. Based on the RFLP data combined with the result of the deletion study, an estimated diagnostic rate for prenatal diagnosis and/or carrier detection in the Japanese DMD families was 63%. The real diagnostic rate obtained from the prenatal and carrier diagnoses, which were practically performed in 17 families, corresponded to the estimation. A protocol useful for the diagnosis in Japanese DMD families is presented.
We have evaluated the short latency somatosensory evoked potentials (SSEPs) following peroneal and posterior tibial nerve stimulation in 27 normal children and adults, and then applied SSEPs examination following peroneal nerve stimulation to 6 children with neurological deficits. Features of the evoked potentials following peroneal nerve stimulation in normal children were almost similar to those in adults, but we found several points characteristic in children; a higher incidence of evoked potentials and a clearer appearance of "standing potential" at the lower thoracic vertebral level than in adults. Spinal afferent conduction velocity reached at a maximum at 3-4 years of age. The SSEPs following peripheral nerve stimulation in lower extremities are useful in pediatric neurology to determine the level of the spinal lesion, to reveal the distribution and pathophysiology of the spinal dysfunction, and to analyze the process of the disease progression.
P4 (P14 in adults), N' (N18) and N1' (the negative wave after cortical N1, obtained until late infancy) of short-latency somatosensory evoked potentials (S-SEPs) by median nerve stimulation were studied in 13 selected patients. Of these, 12 patients were suggested to have brain-stem lesions responsible especially for the cardinal symptoms and at the same time showed abnormal S-SEPs characteristically between P3 (P13) and N1 (N20). Another patient showed no cortical response with multilocular cystic encephalopathy. Each wave component could be identified by the same methodology as in controls from neonates to adults. In the normal subject the wave forms of P4 and N' were slightly influenced by sleep level. The abnormal S-SEP findings could be classified by an increase and decrease in the wave amplitudes or by a shortening and prolongation in the peak latencies. Upon these classifications we tried to delineate the pathophysiology underlying abnormalities of S-SEPs between P3 and N1. In addition, a negative wave after N1, compatible with N1', was identified in a case without cortical response. Evidence suggests that the origins of P4 and N' may include not only the lemniscal pathway but partly also the extra-lemniscal synapses in the brain-stem. The neural source of N1' seems to be partly in the subcortical structures. As for the clinical significance of S-SEPs in children, the features of P4 and N' are influenced both by the distribution of the affected lesions and by the underlying pathophysiology.
Spinal somatosensory evoked responses to peroneal nerve stimulation were examined in 23 control subjects and 8 patients with pathological lesions of the spinal cord or peripheral nerve. In the control subjects, the response was found as a triphasic potential increasing in latency rostrally at the lumbar spinous recording location. Another negative potential following this triphasic potential appeared at the L1 to T10 spinous recording locations, which might reflect synaptic and/or post-synaptic activity in the spinal cord. This negative response then progressively increased in latency rostrally. The spinal conduction velocity was higher at the upper thoracic leads than at the lower leads. Three patients with spinal cord atrophy showed disappearance of the spinal evoked potential at the spinous recording location corresponding to the pathological lesion. However, since the triphasic potential at the lumbar spinous lead was undetectable in the patients with lesions of the peripheral nerve or cauda equina, the spinal cord function could not be estimated well in these patients.
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