Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis

NB176 is a Bacillus thuringiensis mutant derived by λ-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ-irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.     

Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures

Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8°C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 × 10² to 5.6 × 10² bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6°C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 × 10⁴), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 × 10⁴ bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4°C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10⁵ bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 × 10⁷ was observed. V. salmonicida NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with V. salmonicida NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal Aeromonas sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for Vibrio anguillarum but not for V. salmonicida, whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8°C. Increased bacterial numbers in seawater at 4°C result in augmented invasion of bacteria in hemolymph and soft tissues. V. salmonicida, a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.     

Assignment of the dipeptidylpeptidase IV (DPP4) gene to pig Chromosome 15q21

A porcine 2-kb partial dipeptidylpeptidase IV (DPP4, EC 3.4.14.5) cDNA clone and a porcine 16-kb genomic fragment containing parts of the DPP4 gene were isolated, characterized, and used as probes to map the DPP4 gene to pig Chr (Chr) 15q21 by fluorescence in situ hybridization. A two-allele RFLP was revealed for the DPP4 gene. This polymorphism was utilized in a linkage test against the erythrocyte antigen G (EAG), previously assigned to Chr 15, and the microsatellite S0088, which is linked to EAG. The linkage analyses revealed significant evidence for linkage confirming the assignment of DPP4 to Chr 15.     

Etlingera kenyalang (Zhgiberaceae) — a new species from Sarawak, Borneo, with notes on its ecology and Iban ethnobotany

Etlingera kenyalang is described from southern Sarawak. Ecological notes are given including a possible pollinator and seed dispersers. The Iban ethnobotany of the species is discussed; the shoots and fruits are edible and the leaves are used ritually during the Gawai Kenyalang (Hornbill Festival).     

Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm.

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.     

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

Immunocytochemical mapping of gastrin/CCK-like peptides in the neuroendocrine system of the blowfly Calliphora vomitoria (Diptera)

Summary The distribution of gastrin/CCK-like immunoreactive material has been studied in the retrocerebral complex of Calliphora. The material reacts with antisera specific for the common COOH terminus of gastrin and CCK but not with N-terminal antisera. The three thoracic ganglia and the fused abdominal ganglia each contain a specific number of symmetrically arranged immunoreactive cells both dorsally and ventrally in pairs on either side of the midline in a sagittal plane. The neuropil of these ganglia also contains a considerable amount of immunoreactive fibres and droplets. Reconstructed axonal pathways suggest that some of the nerve fibres have their origins within the brain and/or the suboesophageal ganglion. Immunoreactive material may also be seen apparently leaving the thoracic ganglion posteriorly via the abdominal nerves, and there is strong evidence of a neurohaemal organ within the dorsal sheath in the region of the metathoracic and abdominal ganglia. There appears to be a direct correlation between the content of peptidergic material of cells and fibres and the age and diet of the flies. The corpus cardiacum contains COOH-terminal specific gastrin/CCK-like material within the intrinsic cells and in the neuropil. It is present also in the cardiac-recurrent nerve entering the corpus cardiacum anteriorly and in the nerves leaving the gland dorsoposteriorly, the aortic or cardiac nerves. It is not observed, however, in the nerves leaving the corpus cardiacum ventroposteriorly, the so-called oesophageal, gastric or crop-duct nerves. The corpus allatum and the hypocerebral ganglion do not contain immunoreactive material of this type. Gastrin/CCK-like and secretin-like immunoreactive materials appear to co-exist in the cells of the corpus cardiacum and co-existence of gastrin/CCK-like and pancreatic polypeptide like substances occurs within certain cells of the thoracic ganglion.     

X-Linked mental retardation with fragile X. a pedigree showing transmission by apparently unaffected males and partial expression in female carriers

Summary A large family is reported in which mental retardation associated with the fragile site at Xq28 was found. Three normal males seemed to have transmitted the trait through their daughters to affected grandchildren.A total of 19 family members were investigated cytogenetically. Mentally retarded males showed macroorchidism and the fragile X. Three mentally retarded females were found, with the fragile X in a high percentage of cells; in contrast, the obligate carriers showed no or only few cells with the fragile X.