Patched 1 conditional null allele in mice

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function.     

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.     

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

Further localization of the gene for nevoid basal cell carcinoma syndrome (NBCCS) in 15 Australasian families: Linkage and loss of heterozygosity

Nevoid basal cell carcinoma syndrome (NBCCS; basal cell nevus syndrome or Gorlin syndrome) is a cancer-predisposition syndrome characterized by multiple basal cell carcinomas (BCCs) and diverse developmental defects. The gene for NBCCS has been mapped to 9q23.1-q31 in North American and European families. In addition, loss of heterozygosity (LOH) for genetic markers in this region has been detected in sporadic BCCs, indicating that the NBCCS gene is probably a tumor-suppressor gene. In this study we have determined that the NBCCS gene is also linked to this region in Australasian pedigrees and that there is no significant evidence of heterogeneity. We have defined the localization of the gene by multipoint and haplotype analysis of 15 families, using four microsatellite markers. LOH at these loci was detected in 50% of sporadic BCCs, a rate that is significantly higher than that in other skin lesions used as controls.     

Identification of toxic metals in affected algal cells in assays of wastewaters

Chlorella fusca was the test species used in assays of two wastewaters, one characterized by a high concentration of free metal ions, and the other by high concentrations of free metal ions and chlorinated organic compounds. Fine structural damage in cells exposed for six days to the effluents was examined using an electron microscope. In the presence of organic toxicity, the affected cells displayed a characteristically large build-up of starch grains. At high metal concentrations, the chloroplasts were the only organelles altered. Within the chloroplast, there were disruptions of the thylakoidal membranes and enlargement of interthylakoidal spaces. Excess metals, detected by X-ray microanalysis in the polyphosphate (poly-P) bodies of cells where chloroplast damage was observed, were regarded as harmful. X-ray scanning of particulate material in effluent samples is proposed as a useful and low cost addition to surveillance programmes.     

The effect of translation and transcription inhibitors on the synthesis of periplasmic phosphatases in E. coli.

Previous studies by others have indicated that the synthesis of secreted enzymes is unusually sensitive to many translation inhibitors and resistant, for about 30 min, to rifampicin. We have studied the sensitivity of secreted (periplasmic) phosphatases to such inhibitors. Alkaline phosphatase synthesis is more sensitive than total protein synthesis to tetracyclin and spectinomycin, but not to sparsomycin, streptomycin, chloramphenicol, kasugamycin, blasticidin S or thiostrepton; it is slightly more resistant than total protein synthesis to the latter two antibiotics. Acid hexose-phosphatase was also preferentially sensitive to tetracyclin and spectinomycin and also to kasugamycin. beta-galactosidase was also included in the study, as an intracellular enzyme, and was found to be preferentially inhibited ("repressed"), sometimes transiently, by all eight translation inhibitors. This effect did not seem to be mediated through cyclic AMP or guanosine tetraphosphate; the "repression" was still evident in mutants with altered rho factor indicating that it may also not be related to artificial polarity. Synthesis of both periplasmic phosphatases was immediately inhibited by rifampicin. These results differ from those found in previous studies with other organisms and suggest a reappraisal of the usual interpretation of these phenomena.     

Total disappearance of cell proliferation in the lens of a hypophysectomized animal: in vivo and in vitro maintenance of inhibition with reversal by pituitary factors.

Cell cycle kinetics of lens epithelium were studied in frogs following hypophysectomy, and subsequent replacement therapy with bovine growth hormone (bGH). Hypophysectomy led to a complete absence of cells in DNA synthesis and mitosis within 4 weeks. A small number of G2 nuclei was present in all preparations, but the number was much reduced with time after surgery. Occasional preparations with a significant G2 population were encountered. The major block to cycle traverse appeared to be in the G0-G1 segment. Mechanical injury to the lenses of hypophysectomized animals caused the cells to move closer to S without, however, reaching it. The in vivo block to cycle traverse persisted in organ-cultured lenses maintained in medium containing serum of hypophysectomized animals. The block to cycle traverse was reversed when the lenses were cultured in medium containing the serum of hypophysectomized animals that received replacement therapy with bGH. The serum became effective between 1 and 2 days after the star of replacement therapy. The lens epithelial cells from hypophysectomized frogs incur a deficit such that a protracted prereplicative phase is detected when favourable growth conditions are restored.     

The interaction of cytochrome C with phospholipids. A pulse-radiolysis study.

The reactions of the hydrated electron (eaq-), produced during pulse radiolysis, have been used to study the binding of phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), and phosphatidyl inositol (PI) vesicles with horse-heart cytochrome C. An interaction could only be detected between cytochrome C and either PS or PI. An apparent equivalence point in the binding was reached for both phospholipids at a molar ratio of phospholipid : protein of 6 : 1. At this point, the reactivity of (eaq-) towards the cytochrome C was very markedly reduced. Indeed, the rate of disappearance of (eaq-) under such conditions was the same as the rate of eaq- disappearance in triply-distilled water. The inclusion of cholesterol at a molar ratio of 1 : 1 within the phospholipid vesicles changed the stoichiometry of the interaction. Evidence that protonated epsilon-amino groups of lysine residues are involved in the interaction is presented. Possible models for the complexes formed are discussed.