Cas spleen focus-forming virus. II. Further biological and biochemical characterization.

A new isolate of a murine erythroblastosis-inducing spleen focus-forming virus (Cas SFFV), derived from the wild mouse ecotropic murine leukemia virus Cas-Br-M, was further characterized after the production of a nonproducer cell line. When rescued from the nonproducer cells with a helper murine leukemia virus, the Cas SFFV induced rapid splenic enlargement and focus formation when inoculated into adult NFS/N mice. The Cas SFFV nonproducer cell line was also utilized to compare the envelope-related glycoprotein of Cas SFFV with gp52s from three strains of Friend SFFV. Cas SFFV was found to encode a 50,500-dalton glycoprotein (gp50) distinct in size to the envelope-related glycoproteins of the Friend SFFVs. The Cas SFFV was also compared in RNA blot hybridization studies. The genomic viral RNA of Cas SFFV was found to be slightly larger than two polycythemia-inducing strains of Friend SFFV and markedly larger than the anemia-inducing strain. Further comparisons between the SFFVs were made by examining their transforming capabilities in an in vitro erythroid burst assay. The erythroid bursts induced by Cas SFFV and the anemia-inducing strain of Friend SFFV showed similarities in their erythropoietin requirements. This study supports our recent observations that Cas SFFV is biologically similar to the anemia-inducing strain of Friend SFFV yet biochemically distinct from all Friend SFFVs.     

Response of the gill Na+-K+ ATPase activity, succinic dehydrogenase activity and chloride cells to saltwater adaptation in Atlantic salmon, Salmo salar L., parr and smolt

Gill succinic dehydrogenase and Na⁺-K⁺ ATPase activities were stimulated by salt water transfer in parr and smolt. Both activities were preferentially located in the chloride cells. Salt water adaptation induced proliferation and enlargement of the chloride cells. Hypertrophy of the chloride cell system occurred in parr adapted to salt water.     

Tumour induction by activated abl involves tyrosine phosphorylation of the product of the cbl oncogene.

v-cbl is the transforming gene of a murine retrovirus which induces pre-B cell lymphomas and myelogenous leukaemias. It encodes 40 kDa of a gag fusion protein which is localized in the cytoplasm and nucleus of infected cells. The c-cbl oncogene encodes a 120 kDa cytoplasmic protein and its overexpression is not associated with tumorigenesis. The c-cbl sequence has shown that v-cbl was generated by a truncation that removed 60% of the C-terminus. In this study, we carried out experiments to identify the position within cbl where the transition occurs between non-tumorigenic and tumorigenic forms. These experiments focused attention on a region of 17 amino acids which is deleted from cbl in the 70Z/3 pre-B lymphoma due to a splice acceptor site mutation. This mutation activates cbl's tumorigenic potential and induces its tyrosine phosphorylation. We also show that the expression of the v-abl and bcr-abl oncogenes results in the induction of cbl tyrosine phosphorylation, and that abl and cbl associate in vivo. These findings demonstrate that tyrosine-phosphorylated cbl promotes tumorigenesis and that cbl is a downstream target of the bcr-abl and v-abl kinases.     

Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae

In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.     

Abundance, sizes and developmental stages of larval krill, Euphausia superba, during winter in ice-covered seas west of the Antarctic Peninsula

Larval krill were sampled west of the Antarctic Peninsula duringthree winter cruises: September 1991, June 1993 and September1993. Larval abundances were estimated from net catches andcompared directly to visual counts (made by a SCUBA diver) oflarvae occupying the ice habitat at the same sampling stations.The number of larvae per square meter sampled with nets wasmore often greater than that observed by the diver, irrespectiveof the sampling period. However, comparisons of larval abundancewithin sampling periods were not statistically significant.Larval krill collected by divers were significantly larger thanthose collected with nets for each of the three cruises. Thestage composition of larval krill also depended on the collectionmethod: net-collected samples contained a disproportionatelyhigh number of early furcilia larvae in June 1993 (early winter),and a disproportionately low number of early juveniles duringSeptember 1991 and 1993 (late winter). These results lead usto suggest that larval/juvenile krill occupy both the watercolumn and sea ice habitat during the austral winter, and thatthere are often differences in the sizes and developmental stagesof the two groups. For larval krill that occupied the sea icehabitat, aggregations were larger and more numerous during latewinter than in early winter. In addition, larvae within aggregationsoccupied structurally complex microhabitats, provided by over-raftedice floes, more often than they occupied smooth, downward-facingice surfaces where ice was not over-rafted.     

Observations on new Myxobolus species and Kudoa species infecting the nervous system of Australian fishes

Myxobolus galaxii sp. nov. from the spinal cord of Galaxias olidus in northeast Victoria induced little pathologicaf change even when present in lare numbers. Myxobolus gadopsii sp. nov. was found in the meninx and other connective tissues of Gadopsis marmoratus popufations within all three mainland Victorian enotypes as well as in G. bispinosus. Myxobolus gadopsii also appeared to be harmless to its hosts. Kudoa sp. from the brain of Lates calcarifer in north Queensland was associated with abnormal swimming behaviour in juveniles but not in adults. A further Kudoa species, probably K. nova or K. clupeidae, was examined in Thunnus maccoyii from Western Australia, where it was found that most, if not all, of the infections developed in peripheral nerves.     

The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation.

The v-cbl oncogene is the transforming gene of the murine Cas NS-1 retrovirus which induces pre-B cell lymphomas and myeloid leukaemias. Sequencing of c-cbl has revealed that v-cbl was generated by a large truncation that removed 60% of the C-terminus of the corresponding protein. In this study we prepared antibodies to cbl and found that c-cbl encodes a 120 kDa protein which is localized in the cytoplasm with a cytosolic and cytoskeletal distribution. Immunofluorescence studies show a striking pattern of brightly staining vesicles in mitotic cells similar to that observed with cytokeratin antibodies. In contrast to p120c-cbl, which is exclusively cytoplasmic, the p100gag-v-cbl encoded by Cas NS-1 is localized in both the cytoplasm and the nucleus. This redistribution to the nucleus correlates with the ability of cbl to induce acute transformation. Furthermore the truncated protein encoded by v-cbl can bind DNA, unlike the full-length protein. These results suggest that the C-terminus of cbl is involved in the retention of p120c-cbl in the cytoplasm and the inhibition of DNA binding. The findings also suggest that a truncated protein encoded by c-cbl exists in the nucleus of normal cells.     

Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance.

31P nuclear magnetic resonance (NMR) spectra of human low density lipoprotein (LDL) has been obtained and the major phospholipid components identified. Analysis of the spectra revealed two phospholipid environments: one occupied by 4/5 of the phospholipid with high resolution resonances possessing properties similar to phospholipids in vesicles, and a second occupied by 1/5 of the phospholipid with broad lines indicative of immobilization. Limited trypsin treatment of the particle cleaved all of the B peptide into smaller molecular weight peptides which remained with the particle. Trypsin-treated LDL eluted from a Sepharose CL-6B column similarly to native LDL so that the modified particle remained intact. 31P NMR spectra of trypsin-treated LDL showed little or no immobilized phospholipid. The immobilization in the native LDL particle is attributed to lipid-protein interactions between 1/5 of the phospholipid and the B peptide.     

Catalytic domains of tyrosine kinases determine the phosphorylation sites within c-Cbl

Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates. Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed. Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl. We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-Cbl and CrkL or phosphatidylinositol 3'-kinase (PI3-K). Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins.