Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tailbud cells and zebrafish embryo.

Caspase-3 was cloned from zebrafish embryos and its properties were characterized to identify the biological implications of caspase in embryogenesis and apoptosis in zebrafish, which is a model organism in vertebrate developmental biology and genetics. The predicted amino acid sequence, totalling 282 amino acid residues, consisted of the prodomain and large and small subunits. Phylogenetic analysis showed that the cloned zebrafish caspase was a member of the caspase-3 subfamily with approx. 60% identity with caspase-3 from Xenopus, chicken and mammals. In addition, recombinant zebrafish caspase hydrolysed acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide, and exhibited similar substrate specificity to the mammalian caspase-3 subfamily. Therefore this caspase was designated zebrafish caspase-3. Overexpression of zebrafish caspase-3 induced apoptosis and increased ceramide levels in fish fathead minnow tailbud cells and zebrafish embryos. Both ceramide generation and apoptosis induction were inhibited by treatment with a caspase inhibitor, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone. Moreover, zebrafish caspase-3 mRNA was present in early embryos up to the 1000-cell stage as a maternal factor, and was then expressed throughout the body after the gastrula stage by zygotic expression. These findings indicate that the isolated caspase-3 plays an important role in the induction of ceramide generation as well as apoptosis in fish cells and the zebrafish embryo, and suggest that caspase-3 functions as a modulator of the pro-apoptotic signal in development.

Synthesis of NG-061 and its analogs, and their biological evaluation as an enhancer of nerve growth factor.

A novel potentiator of nerve growth factor (NGF), NG-061, which had been isolated from the fermentation broth of Penicillium minioluteum F-4627, was synthesized from methoxybenzoquinone and phenylacetylhydrazine in a single step. A series of acyl hydrazone derivatives were also synthesized and their potentiator activity of neurotrophic effect of NGF on neurite outgrowth was evaluated by assay with a rat pheochromocytoma cell line PC12.

A case of human adjuvant disease after augmentation rhinoplasty.

Human adjuvant disease (HAD) is an autoimmune syndrome which is caused by prolonged hypersensitization of injected foreign materials. Usually, this occurs after mammary augmentation with foreign materials. We report a rare case of HAD after rhinoplasty with silicone injection. Thirty years ago, the patient underwent augmentation rhinoplasty with silicone injection. We removed the silicone and grafted the area with fascia lata. After the operation, local and systemic symptoms improved.

Supplement of liver enzyme by intestinal and kidney transplants in congenitally enzyme-deficient rat.

Gunn rats have a congenital deficiency of bilirubin-uridine diphosphate glucuronyltransferase (B-UDP-GT) activity and are unable to glucuronidate bilirubin in the bile, resulting in unconjugated hyperbilirubinemia. Other than the liver, several organs, including small bowel and kidneys, are known to have B-UDP-GT activity in normal rats. We performed total- or partial-small-bowel transplantation as well as kidney transplantation for Gunn rats in congenic combination and compared the effects of these procedures. Serum total bilirubin (TBil) levels significantly decreased from 7.84 +/- 0.24 mg/dl to 2.19 +/- 0.43 mg/dl 2 weeks after total-small-bowel transplantation (n = 12). Correlation of hyperbilirubinemia was roughly proportional to the length of the transplanted small bowel. There were no difference in metabolic correction between jejunal and ileal transplantation. Serum TBil levels significantly decreased from 7.83 +/- 0.21 mg/dl to 2.24 +/- 0.98 mg/dl 2 weeks after kidney transplantation (n = 5). In conclusion, small-bowel and kidney transplantation were effective in correcting metabolic abnormality in Gunn rats for the period of 4-6 months. Estimated total B-UDP-GT activity supplemented by small-bowel or kidney transplantation was about 1/5-1/4 of the minimal requirement for the complete normalization of serum total bilirubin levels.

Interaction of transcription factor YY1 with a replication-enhancing element, REE1, in an autonomously replicating human chromosome fragment.

We have previously shown that autonomous replication of human chromosome fragments is stimulated by the presence of an 18 bp sequence, REE1, which exhibits transcriptional silencer activity. The REE1 sequence is partly homologous with the serum response element (SRE) required for expression of the human c- fos gene. Here we have examined interaction of REE1 with human nuclear proteins using a gel retardation assay. One of the REE1-protein complexes formed showed almost the same mobility as the SRE-protein complex and complex formation was competitively inhibited by the SRE fragment. The protein complex with REE1 as well as that with SRE was found to contain the transcription factor YY1, known to bind to the SRE. These results suggest that YY1 protein may participate in stimulation of replication through its interaction with REE1.

Leukoencephalopathy following treatment with carmofur: a case report and review of the Japanese literature.

A 53-year-old woman was treated with 5 courses of CAP treatment following operation for FIGO Stage Ia cancer of the ovary in September 1986. And in April 1987, she started an oral adjuvant chemotherapy with 400 mg/day of carmofur. In early June, she developed vertigo and dysarthia and was hospitalized. A CT scan showed low-density areas adjacent to both lateral ventricles, and an EEG revealed abnormally slow waves. She improved gradually after carmofur was discontinued and left the hospital in October 1987. There have been 24 reported cases of leukoencephalopathy because of carmofur in Japan, but the pathophysiological mechanism involved is not known. Since it is more common in women than in men, its incidence will probably increase in gynecological patients. Therefore, we must be on the lookout for central nervous system signs and symptoms in patients receiving adjuvant chemotherapy with carmofur.

[Bacteriological and clinical study of ciprofloxacin in gonococcal urethritis].

The purpose of this study was to determine the susceptibility of Neisseria gonorrhoeae to ciprofloxacin (CPFX). The MIC's of CPFX against 50 clinical isolates of N. gonorrhoeae was examined and they were between less than or equal to 0.003 microgram/ml and 0.006 microgram/ml including 8 beta-lactamase producing strains. The CPFX was administered orally to 3 groups of 10 cases with gonococcal urethritis, groups being determined by 3 dose levels: a group with 200 mg b.i.d. for 3 days (a total of 1,200 mg), another with 400 mg b.i.d. (a total of 800 mg) and the final group with single administration of 400 mg. The effect of CPFX in the 1,200 mg-administered group was excellent in 3 and good in 7. The effect of the drug in the 800 mg-administered group was excellent in 2 and good in 8. The effect in the 400 mg-administered group was all good. Therefore, the overall cure rate was 100% including 5 patients with beta-lactamase producing gonococcic infection. Side effects were not observed in the 30 cases.

Phosphatidylinositol-specific phospholipase C in fetal membranes and uterine decidua.

An assay procedure was developed in which phosphatidyl[2-(3)H]inositol was employed as substrate for the measurement of phosphatidylinositol-specific phospholipase C activity. Employing this assay, phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua was identified and characterized. The specific activity of this enzyme in amnion (4.4 mumol x mg(-1) protein x h(-1)) was three times that in uterine decidua and more than five times that in chorion laeve. No difference was found between the specific activity of phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The products of phosphatidylinositol hydrolysis in short-term incubations were stoichiometric amounts of diacylglycerol and inositol-1,2-cyclic-phosphate plus inositol-1-phosphate. After longer periods of incubation, monoacylglycerol also was detected. Diacylglycerol lipase activity also was demonstrated in these tissues. More than 90% of phosphatidylinositol-specific phospholipase C activity of amnion tissue was recovered in the 105,000-g supernatant fraction, and optimal enzymatic activity in vitro was observed at pH 6.5-7.5 in the presence of Ca(2+) (8 mM) and mercaptoethanol (4 mM). Phosphatidylinositol-specific phospholipase C activity was stimulated by fatty acids in low concentrations, but was inhibited by lysophosphatidylcholine and a variety of detergents. No effect of labor on the specific activity of phosphatidylinositol-specific phospholipase C in either fetal membranes or uterine decidua could be detected. The finding of an active phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua is complementary to our previous finding of a selective loss of arachidonic acid from phosphatidylinositol of human fetal membranes during labor. The action of phosphatidylinositol-specific phospholipase C, coupled to diacylglycerol lipase action, could provide a mechanism for the release of arachidonic acid for prostaglandin biosynthesis during parturition.

Histamine release from human leukocytes: modulation by cadmium ion.

The effect of cadmium on histamine release was studied in relation to the role of calcium. The antigenic histamine release from peripheral leukocytes of ragweed-sensitive patients was inhibitied in the presence of cadmium (greater than 10(-5) M). Spontaneous histamine release was not affected by cadmium except for an enhancement observed in high concentrations (greater than or equal to 10(-3)M). Cadmium did not seem to affect the first stage of histamine release but to act mainly on the calcium-dependent second stage. Increasing the level of calcium in the medium could antagonize the inhibitory effect of cadmium. The effect of cadmium could be totally abolished by deuterium oxide and partially by dibutyryl cyclic adenosine monophosphate (AMP) in certain concentrations. These results would indicate that: (1) cadmium acts as an antagonist of calcium and can be used for the study of the role of calcium in histamine release, (2) the action of calcium in the histamine release reaction seems to be related to the microtubular system, (3) cyclic AMP may potentiate histamine release when the action of calcium is inhibited.

The effect of Tris-buffered media and Tyrode physiologic saline solution on the antigenic release of histamine from human leukocytes.

Allergic histamine release from leukocytes was compared in three different media: Tyrode physiologic saline solution, Tris-buffered saline containing human albumin, calcium, and magnesium (Tris-ACM), and Tris-ACM with homologous serum. In a selected group of low histamine releasers, the maximal amount of antigenic histamine release was significantly higher in Tyrode solution as compared to Tris-ACM buffer. When homologous serum was added to Tris-acm, an enhancement of histamine release greater than with Tyrode solution was obtained. These results suggest that Tris-ACM may not be the optimal buffer for leukocyte histamine release experiments. Since Tyrode solution contains no serum proteins that bind slow-reacting substance of anaphylaxis or prostaglandins, the use of this medium may be advantageous for the study of the release of the chemical mediators from human leukocytes.