Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila

The temperature-sensitive Drosophila mutant, shibirets1 (shi), has been shown to exhibit a reversible block in synaptic transmission at 30 degrees C. Various synaptic terminals (neuromuscular, sensory, central) of this mutant were observed by electron microscopy. At 19 degrees C, all terminals of shi showed essentially the same structural features as those of wild-type (Oregon-R) flies, while at 30 degrees C (5 or 10 min of exposure) shi terminals exhibited various structural changes not seen in the wild type. The major structural change observed in all of the various types of terminals was the accumulation of many pitlike structures on the plasma membrane near presynaptic sites. These structures consisted of a spherical head portion, about 50-100 nm in diameter, and a cylindrical neck portion, about 20 nm long and 20-25 nm in diameter. The neck portion was surrounded by a kind of cytoplasmic dense material, about 10 nm thick, reminiscent of a "collar." Thus, these pits are referred to as "collared pits." Similar kinds of pits were observed, although very rarely, in wild-type flies at 19 and 30 degrees C and in shi flies at 19 degrees C. In addition, various degrees of vesicle depletion, and an increase in membranous structures (infoldings and cisternalike or tubulelike structures) often accompanied pit formation. The possibility that these pits are the result of a blocked step in the endocytotic process, which in turn causes vesicle depletion as exocytosis proceeds, is discussed.     

Hepatic microsomal conversion of pregnenolone to 3β,5,6β-trihydroxy-5α-pregnan-20-one via pregnenolone α- and β-epoxides

In the presence of ferrous ion, ADP, and an NADPH-generating system, [4-¹⁴C]pregnenolone was oxidized by bovine liver microsomes to its α-epoxide (5,6α-epoxy-3β-hydroxy-5α-pregnan-20-one), β-epoxide (5,6β-epoxy-3β-hydroxy-5β-pregnan-20-one), trihydroxypregnanone (3β,5,6β-trihydroxy-5β-pregnan-20-one) which were separated, isolated on an octadecylsilicone column in 70% aq. methanol by high performance liquid chromatography, identified with respective synthetic specimens by gas-liquid chromatography-mass spectrometry. The microsomal Δ⁵-oxidation products of pregnenolone were detected in trace yield either when EDTA was added to the incubation mixture or when ferrous ion was omitted from the mixture. The microsomal oxidation system generated malondialdehyde significantly. It, however, was retarded to a negligible extent either by the addition of EDTA or by the omission of ferrous ion. Therefore, the microsomal formation of the significant yields of Δ⁵-oxygenated pregnenolones was reasonably attributed to a reaction linked to microsomal lipid peroxidation. The ratio of pregnenolone α- to β-epoxides formed was 1:3. A comparable study carried out under the same conditions by using [4-¹⁴C]cholesterol as the substrate resulted in the similar Δ⁵-epoxidation with concomitant formation of cholestane-3β,5α,6β-triol; cholesterol α- and β-epoxides formed were in the ratio 1:4.Both pregnenolone α- and β-epoxides were hydrolyzed by the microsomes to trihydroxypregnanone as the sole metabolite at a relative rate of 0.6:1. A similar relative value was also obtained in the microsomal hydrolysis of cholesterol α- and β-epoxides to the cholestanetriol.     

Detection of nitric oxide production in lipopolysaccharide-treated rats by ESR using carbon monoxide hemoglobin.

Release of nitric oxide (NO), from macrophages activated with E. coli lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO2-. The present study was aimed to prove in vivo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. NG-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO2- and HbCO. HbNO was, therefore, derived from NO, not from NO2-. These results show that free NO is produced in vivo by the stimulation of LPS.     

Relation of proliferative activity to survival in patients with advanced gastric cancer

The proliferative activities of 98 biopsied specimens of advanced gastric cancers were measured by the in vitro bromodeoxyuridine (BrdU) labelling method. BrdU labelling indices (LIs) varied from 4.8 to 45.1%. There was no correlation of BrdU LIs and histological type, distant metastasis, serosal invasion, macroscopic type or tumor size. However, BrdU LIs of tumors with lymph node metastases were significantly higher than those without them. Patients with tumors showing high BrdU LIs had a three-fold relative risk of death, as compared with those showing low BrdU LIs. When the BRdU LIs and all the clinicopathological parameters were entered simultaneously into the Cox model, BrdU LIs, peritoneal dissemination and serosal invasion emerged as independent prognostic parameters. These results indicate that the in vitro BrdU labelling method using biopsied materials may be useful in predicting lymph node metastasis preoperatively and designing operative procedures for individual patients.     

Effects of ketamine on the cholecystokinin,somatostatin, substance P,and thyrotropin releasing hormone in discrete regions of rat brain

Intraperitoneal injection of ketamine (100 mg/kg body weight) significantly reduces the levels of cholecystokinin (CCK) somatostatin (SRIF), and substance P (SP)-like immunoreactivity in various regions of rat brain. No significant change in thyrotropin releasing hormone (TRH)-like immunoreactivity was observed. Neuropeptide systems may be involved in the neuropharmacologic effects of ketamine.     

Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin

A cDNA encoding a human endothelium-derived vasoconstrictor peptide, endothelin, was isolated from a human placenta cDNA library. The nucleotide sequence of this cDNA clone showed that the primary structure of the human preproendothelin has 212 amino acid residues and is highly homologous to porcine preproendothelin, and that human endothelin is identical with porcine endothelin.     

Isolation of mutants showing temperature-sensitive cell growth from embryonal carcinoma cells: control of stem cell differentiation by incubation temperatures

Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.     

Contraction and intracellular calcium-ion elevation of cultured human aortic smooth muscle cells by endothelin-1, vasoactive intestinal contractor (VIC) and the derivatives

Summary Effects of endothelin (ET) family peptides and their derivatives on cellular contraction and calcium-ion level were examined by using cultured human vascular smooth muscle cells (VSM). Contraction of cultured human VSM, isolated from human fetal aortic segments, was induced within 1 min after the treatment with ET-1 (100 nM) as seen in the changes of cytosolic calcium-ion localization. In parallel with the cell contraction, cytosolic calcium-ion level in the human VSM increased very rapidly and then dropped with some oscillation as determined by Anchorage Cell Analyzing System. It was noted that transient calcium-ion mobilization rather than sustained calcium-ion influx was significant in the contraction of cultured human VSM. Vasoactive intestinal contractor (VIC), three amino acids different from ET-1, had less activity in increase of intracellular calcium-ion level and in percent of response cells than ET-1, ET-2, and VIC-S4L6 (one amino acid different from ET-1). EC₅₀ of ET-1, VIC-S4L6, ET-2, and VIC were 0.5 nM, 0.6 nM, 2.0 nM, and 20 nM, respectively. VIC-like peptide (VIC-LP), 16 amino acids fragment of VIC precursor protein, had no effect with a single administration of up to 10 μM. However, the increase in calcium-ion level by VIC was suppressed with a prior treatment of cells with high concentration (10 μM) of VIC-LP. The establishment of cultured human VSM for the simultaneous examination of the contraction and calcium-ion level will provide a new system to study signal transduction of vasocontractor peptides.