Desensitization of CholecystokininB Receptors in GH3 Cells

Abstract: Desensitization of the cholecystokinin (CCK) octapeptide (CCK-8)-induced rise in intracellular free calcium concentration ([Ca²⁺],) was characterized in GH₃ cells, a pituitary tumor cell line, which are known to possess CCK_B receptor subtype. The CCK-8-induced [Ca²⁺], transient was reduced following the initial application of CCK-8. A similar desensitization of the CCK-8-induced response was observed following the first application of thyrotropin-releasing hormone (TRH). By contrast, the TRH- induced response was not desensitized by the preceding application of CCK-8. Desensitization of the CCK-8-induced [Ca²⁺], transient was associated with diminished inositol 1,4,5-trisphosphate formation. The recovery of desensitization of the CCK-8-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A (100 n M ). The responsiveness to CCK-8 was also reduced by phorbol 12, 13-dibutyrate (PDBu), and this effect of PDBu was completely abolished by preincubation with staurosporine. Staurosporine significantly attenuated the desensitization caused by preincubation with CCK-8, but this effect was too small to attribute the desensitization to the protein kinase C transduction pathway alone. It is likely that desensitization of CCK receptors involves multiple transduction pathways.     

Mucopolysaccharidosis type II (Hunter disease): 13 gene mutations in 52 Japanese patients and carrier detection in four families

Southern blot analysis of the iduronate sulfatase (IDS) gene in 52 unrelated Japanese patients with mucopolysaccharidosis type II was carried out using a cDNA probe, and mutations in 13 patients (25%) were identified. Of these, 3 had partial gene deletions (in 2 the normal 9.4-kb fragment was absent and in 1 the normal 7.4-kb fragment was absent, as determined by Southern blot analysis using EcoRI-digested DNA, respectively), 2 had gene insertions (in 1 there was a unique 11.2kb fragment and in the other there was a unique 5kb fragment, determined by Southern blot analysis using EcoRI digested DNA), and 8 had rearrangements (in 6 the normal 9.4kb and 7.0kb fragments were absent and a unique 11.2kb fragment was present; in the remaining 2 patients there were different rearrangements). In these 13 patients, the similar Southern blot patterns were indicative of structural alterations of the IDS gene, as revealed when their DNA was digested with HindIII or PstI and probed with IDS cDNA. All patients with these structural alterations were in a clinically severe state, except for 1 with an intermediate clinical phenotype. Our analyses of four families among those of the 13 patients revealed that all four mothers were carriers. The detection of structural abnormalities led to a precise identification of Hunter heterozygotes and revealed one de novo rearrangement in a germ cell of one of the maternal grandparents.     

Identification and Characterization of Heat-Stable Enterotoxin II-Producing Escherichia coli from Patients with Diarrhea

Stock strains of Eschericia coli isolated from patients with traveller's diarrhea were examined for production of heat-stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti-STII antiserum. These results suggest that STII-produciing enterotoxigenic E. coli can cause human diarrhea.     

Mucopolysaccharidosis IV A: Assignment of the Human N-Acetylgalactosamine-6-Sulfate Sulfatase (GALNS) Gene to Chromosome 16q24

Plasmid clones of three independent genomic fragments of the gene for human N -acetylgalactosamine-6-sulfate sulfatase (GALNS; EC 3.1.6.4) were utilized in a fluorescence in situ suppression hybridization study to assign the locus to chromosome 16q24. Enzyme assay for GALNS in a patient with del(16)(q22.1) confirmed this finding.     

Novel Subtype of Peroxisomal Acyl-CoA Oxidase Deficiency and Bifunctional Enzyme Deficiency with Detectable Enzyme Protein: Identification by Means of Complementation Analysis

We describe four infants with a novel subtype of an isolated deficiency of one of the peroxisomal β-oxidation enzymes with detectable enzyme protein. The patients showed characteristic clinical and biochemical abnormalities, including hypotonia, psychomotor retardation, hepatomegaly, typical facial appearance, accumulation of very-long-chain fatty acids, and decreased lignoceric acid oxidation. However, β-oxidation enzyme proteins were detected by immunoblot analyses, and large peroxisomes were identified by immunofluorescence staining. In order to identify the underlying defect in these patients, complementation analysis was introduced using fibroblasts from these patients and patients with an established deficiency of either acyl-CoA oxidase or bifunctional enzyme, as identified by immunoblotting. In the complementing combinations, fused cells showed increased lignoceric acid oxidation, resistance against 1-pyrene dodecanoic acid/UV selection, and normalization of the size and the distribution of peroxisomes. The results indicate that two patients with a more severe clinical course were suffering from bifunctional enzyme deficiency and that the other two infants, who were siblings and had a less severe clinical presentation, were the first patients with acyl-CoA oxidase deficiency with detectable enzyme protein.     

Fossil wood flora of the early Miocene Nawamata formation of Monzen,Noto Peninsula,central Japan

Silicified wood collected from the Lower Miocene Nawamata Formation at two localities, Nakaya and Nigoriike, Monzen-machi, Noto Peninsula, central Japan, were identified. Among the 58 specimens there are two species of conifers and eleven species of dicotyledons:Taxodioxylon cunninghamioides (Watari) Watari andT. sequoianum (Merckl.) Gothan (both Taxodiaceae),Carya protojaponica Watari (Juglandaceae),Pterocarya rhoifolia Siebold et Zucc. (Juglandaceae),Ostrya monzenensis sp. nov. (Betulaceae),Quercus anataiensis (Watari) Watari (Fagaceae),Liquidambar hisauchii comb. nov. (Hamamelidaceae),Prunus iwatense (Watari) Takahashi et Suzuki (Rosaceae),Gleditsia paleojaponica comb. nov. (Leguminosae),Acer watarianum Takahashi et Suzuki (Aceraceae),Meliosma mio-oldhami sp. nov. (Sabiaceae),Reevesia miocenica Watari (Sterculiaceae), andFraxinus notoensis sp. nov. (Oleaceae). The fossil wood floras at the two localities are compared to the Daijima Flora, and warm-and/or cool-temperate mesic forests are suggested to occur in the Early Miocene of Monzen.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Microfibrillar structure of growing cell wall in a coenocytic green alga,Boergesenia forbesii

A fine structure of cell wall lamellae in a coenocytic green algaBoergesenia forbesii was examined by electron microscopy. The wall has a polylamellate structure containing cellulose microfibrils 25 to 30 nm in diameter. The outer surface of the cell was covered by a thin structureless lamella, underneath which existed a lamella containing randomly-oriented microfibrils. The major part of the wall consisted of two types of lamellae, multifibrillar lamella and a transitional, matrix-rich one. In the former, microfibrils were densely arranged more or less parallel with each other. In the transitional lamella, existing between the multifibrillar ones, the microfibril orientation shifted about 30° within the layer. The fibril orientation also shifted 30° between adjacent transitional and multifibrillar layers, and consequently the microfibril orientation in the neighboring multifibrillar layers shifted 90°. It was concluded that the orientation rotated counterclockwise when observed from inside the cell. Each lamella in the thallus wall become thinner with cell expansion, but no reorientation of microfibrils in the outer old layers was observed. In the rhizoid, the outer lamellae sloughed off with the tip growth.