Ken Higashi

Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus

The nucleotide sequences of genes encoding the thermostable direct (TSD) hemolysin and the thermolabile (TL) hemolysin of Vibrio parahaemolyticus were determined. From the nucleotide sequence of the TSD hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 189 amino acids and 165 amino acids, and that the molecular weights were 21.1 kDa or 18.5 kDa, respectively. Our data regarding TSD hemolysin were in complete agreement with previously published data. From the nucleotide sequence of the TL hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 418 amino acids and 398 amino acids, and that the molecular weights were 47.5 kDa and 45.3 kDa, respectively. The GC content of the TSD hemolysin gene was 35.6%, while that of the TL hemolysin gene was 47.6% which is almost the same as that of V. parahaemolyticus genome. Maxicell analysis revealed that the molecular weights of the proteins encoded by the TSD hemolysin gene were 22.0 and 19.5 kDa, and that of the protein encoded by the TL hemolysin gene was 45.5 kDa, and that the promoters of these two hemolysin genes of V. parahaemolyticus were functional in Escherichia coli.

Analysis of N-myc amplification in relation to disease stage and histologic types in human neuroblastomas

Both untreated and treated primary neuroblastomas from 52 patients were analyzed to determine the correlation between the amplification of N‐myc oncogene and various prognostic factors. Amplification of N‐myc was observed in eight of 28 untreated cases and in 12 of 24 treated cases. As a whole, 12 of 18 tumors (67%) in Stage IV had N‐myc amplification, but there were fewer cases in the unadvanced disease stage, as reported previously by others. Furthermore, the authors detected N‐myc amplification in three of nine tumors in Stage IV‐S, although the amplification was less than 50 copies. Analysis of progression‐free survival at 24 months revealed that amplification of N‐myc was associated with the worst prognosis (P < 0.001). In the untreated group, no amplification of N‐myc was detected in any of two ganglioneuromas and four ganglioneuroblastomas, whereas amplification of N‐myc was observed in all two round‐cell and six of 20 rosette fibrillary neuroblastomas. On the other hand, the authors detected amplification of N‐myc in three of eight less differentiated ganglioneuroblastomas in the treated group and observed the worst prognosis in these three patients. The total percentage of the cases from both untreated and treated groups suggest that amplification of N‐myc may occur more frequently in undifferentiated types of neuroblastomas than in less malignant types. In conclusion, the amplification of N‐myc in neuroblastomas was closely associated with the worst prognosis, which was suggested by both disease stage and histologic characteristics.

N-myc oncogene amplification and prognostic factorsof neuroblastoma in children

The N-myc oncogene of 28 neuroblastic tumors obtained from 16 untreated and 12 pretreated children was clinically evaluated and compared with known prognostic factors. Significant amplification of the N-myc (more than ten copies) was observed in 0 of 2 tumors in stage I, 1 of 5 in stage II, 2 of 6 in stage III, 6 of 9 in stage IV, and 2 of 6 in stage IVS. In stages II, III, IV, and IVS, all 15 patients with low N-myc amplification (under ten copies) are alive without disease, while among 11 patients with the amplification, seven died with progressive disease and two have a recurrence (P less than .01). All tumors with N-myc amplification originated from the suprarenal region and the amplification appeared in 55% of those from that origin. The amplification also correlated with the age factor. These results suggest that the genomic amplification of N-myc seems to be correlated with known prognostic factors of neuroblastoma, and may be a reliable factor even in the case of preoperatively treated tumors.

Selective suppression of nucleolar RNA metabolism in the absence of protein synthesis

Abstract Ribosomal proteins are required for the maturation of ribosomes from newly formed ribosomal RNA. The fate of newly synthesized ribosomal RNA in the nuclei of regenerating rat liver was studied after the administration of cycloheximide. Remarkable suppression of transport of ribosomes from the nucleus to the cytoplasm was observed in the absence of protein synthesis. Furthermore, it was found that 3 h after cycloheximide treatment the 32 P-labelled RNA which remained in the nuclei did not show the characteristic [ 32 P]base composition of ribosomal RNA. (The A+U G+C ratio of nuclear RNA increased from 0.78 to 1.01 under these conditions.) Both the ultraviolet and the radioactivity patterns of sedimentation profiles of nuclear RNA showed lowered 45-S RNA after cycloheximide treatment. Since the nucleolus is the main site of ribosomal RNA biosynthesis, the rates of [ 14 C]orotic acid incorporation into nucleoli and into the extranucleolar region in nuclei were compared and it was observed that in the presence of cycloheximide nucleolar RNA formation was suppressed to 60% of the control value. These results suggest that protein synthesis is required for both continual formation of ribosomal RNA and for maturation of ribosomes.

Amplification of N-myc oncogene in stage II and IVS neuroblastomas may be a prognostic indicator

The amplification of N-myc oncogene is frequently observed in tumors of patients with advanced, poor prognostic stages of neuroblastoma (unfavorable stage group). However, there has been little documentation of the N-myc amplification in tumors of patients with stages II and IVS neuroblastoma (favorable stage group). In this communication, we present data on one patient in stage II and two in stage IVS. In these children, the primary tumors had an amplified N-myc and the clinical course was poor. In addition, from an analysis of the N-myc oncogene of 26 neuroblastomas, genomic amplification of more than three copies was observed in 4 of 11 (36%) in the favorable stage group, and in 9 of 15 (60%) in the unfavorable stage group. Death occurred only in those with an amplified N-myc, in both groups. The smaller copy number of N-myc amplification of stage IVS tumors as compared with those of stages III and IV, appeared to be characteristic. These findings suggest that N-myc amplification may be a reliable prognostic indicator even in the favorable stage group of neuroblastomas, and may be clinically useful as a guide for treating those with a poor prognosis in stages II and IVS.

Induction of heat shock 70 mRNA by cadmium is mediated by glutathione suppressive and non-suppressive triggers.

The induction mechanism of heat shock 70 (Hsp70) gene by cadmium was investigated. In human amniotic WISH cells, Hsp 70 was induced by cadmium in a dose- and time-dependent manner. Cadmium-induced Hsp70 mRNA levels were enhanced 3- to 4-fold after depletion of intracellular glutathione (GSH) by either diethylmaleate or buthionine sulfoximine. Under these conditions, hydrogen peroxide might increase in the absence of substrate for glutathione peroxidase. We found that exogenous hydrogen peroxide alone induced Hsp70 which was further enhanced significantly after GSH-depletion by diethylmaleate. On the other hand, treatment of cells by diethyldithiocarbamate, an inhibitor of superoxide dismutase, induced Hsp70 2-fold over the level of control. This induction was further stimulated by cadmium even in the presence of GSH. Furthermore, a 4-fold increase of intracellular GSH by the treatment of cells with glutathione isopropyl ester did not diminish the cadmium-induced Hsp70. Gel mobility shift assays of nuclear extracts, from these differently treated cells, with oligonucleotide containing a promoter region of Hsp70 gene revealed that the levels of Hsp70 mRNA observed in the present study corresponded to the changes of transcription. These results imply that the induction of Hsp70 mRNA by cadmium is mediated at least partly via reactive oxygen species and attenuated by cellular GSH and that some part of cadmium-induced Hsp70 can not be eliminated by GSH, suggesting that multiple signals are functioning for this induction.

Assembly of the α-subunit of Torpedo californica Na+/K+-ATPase with its pre-existing β-subunit in Xenopus oocytes

Abstract The α- and β-subunits of Torpedo californica Na+/K+-ATPase were expressed in turn in single oocytes by alternately microinjection the specific mRNAs for the α- and β-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing β-subunit, which had been synthesized by injecting mRNA for the β-subunit, could assemble with the α-subunit expressed later in the single oocytes and the resulting αβ complex acquired both ouabain-binding and Na+/K+-ATPase activities. On the other hand, formation of the αβ complex was not detected when the α-subunit was expressed first, followed by the β-subunit. These data suggest that the β-subunit acts as a receptor or a stabilizer for the α-subunit upon the biogenesis of Na+/K+-ATPase.

Injection of antisense oligos to nNOS into nucleus tractus solitarii increases blood pressure.

We investigated the cardiovascular effects of bilateral microinjection of antisense oligodeoxynucleotides (oligos) into the nucleus tractus solitarii (NTS) to neuronal nitric oxide synthase (nNOS) to suppress the expression of nNOS molecular biologically. In urethane-anesthetized, paralyzed Wistar-Kyoto rats, bilateral microinjection of nNOS antisense oligos (20 pmol in a 50nl volume) into the NTS produced a significant increase in mean arterial blood pressure at 30-60min after injection, compared with rats injected with nNOS sense or scrambled oligos. Immunohistochemical study demonstrated that nNOS immunoreactivity in the rat NTS was suppressed by nNOS antisense oligos. These results indicate that suppression of the nNOS gene using antisense in the NTS increases blood pressure.

Isolation of immunochemically distinct form of cytochrome P-450 from microsomes of tulip bulbs.

A highly purified cytochrome P-450 was obtained from the microsomes of tulip bulbs (Tulipa gesneriana L.). The molecular weight (Mr = 52,500) and amino acid composition of this plant cytochrome P-450 are similar to those reported for rat livers. On the contrary, Ouchterlony double diffusion analyses indicated that cytochrome P-450 isolated from tulip bulbs shares no common antigenic determinants with those of 9 other plants, in spite of the presence of comparable contents of cytochrome P-450 and/or trans-cinnamate 4-monooxygenase with tulip bulbs.

Simultaneous activation of heat shock protein (hsp 70) and nucleolin genes during in vivo and in vitro prereplicative stages of rat hepatocytes

Rapidly growing cells usually have high levels of ribosome biogenesis. The sequential expression of protooncogenes during the transition of quiescent hepatocytes to the replicative stage was assumed to be followed by activation of cellular genes related to cell growth such as ribosome biosynthesis. First, the expression of major nucleolar protein (nucleolin or C23) and major heat-shock protein (hsp 70) genes was examined during rat liver regeneration. hsp 70 may function in cell growth and has a characteristic nucleolar location after heat shock. Both nucleolin and hsp 70 mRNA began to increase simultaneously after peaks of c-fos and c-myc, showed a peak 6 h after partial hepatectomy, and declined to the control levels around 20 h. That is, the peaks of nucleolin and hsp 70 mRNA precede the peak of ribosome formation (12-20 h) and DNA replication (24 h). Second, the behavior of nucleolin and hsp 70 mRNA was examined in primary cultured hepatocytes during their G0-G1 transition. Although the amounts of c-myc mRNA reached a plateau around 20 h after the initiation of culture and remained at these levels, DNA synthesis has never been found to start without the addition of EGF and insulin to this system. Both nucleolin and hsp 70 mRNA began to increase at around 20 h (prereplicative stage) and simultaneously decreased in inverse proportion to DNA synthesis induced by these growth factors. Thus, it is possible that the simultaneous enhancement of nucleolin and hsp 70 genes as described above is not merely coincidental, but is important biologically during the transition of quiescent hepatocytes to proliferative cells.