Transformation of Murine Cells by Two “Slow Viruses,” Visna Virus and Progressive Pneumonia Virus

Visna and progressive pneumonia virus (PPV), two antigenically related, non-oncogenic "slow viruses" which have ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, were examined for their ability to transform cells. Murine cells which had been exposed to either visna or PPV developed foci of altered, spindle-shaped cells 3 to 4 weeks after infection. Visna and PPV transformed lines were established from these cultures. There was no evidence that other oncogenic DNA or RNA viruses were involved in the observed transformation. Visna or PPV could be "rescued" from all transformed lines by co-cultivation with normal sheep testis cells. "Rescued" virus was identified as visna or PPV, and they retained the capacity to transform mouse cells. These experiments may have important implications in the understanding of both viral carcinogenesis and "slow" viral infections.     

Conformational feature of neuroactive domoic acid: X-ray structural comparison with isodomoic acid A and alpha-kainic acid.

As an aid for developing a new type of potent insecticide acting on the neuromuscular junction, conformational characteristics of domoic acid and isodomoic acid A, the naturally occurring glutamate agonists, were investigated by X-ray crystal analyses. Conformational comparison with a neuroactive alpha-kainic acid provides information concerning the stereochemical feature responsible for the biological activity.     

Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.     

Association of actin with alpha crystallins

The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.     

Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene.

The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helix-turn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membrane-associated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the phi (nfxB'-lacZ+) (Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.     

Persistent BK papovavirus infection of transformed human fetal brain cells. I. Episomal viral DNA in cloned lines deficient in T-antigen expression.

After infection of permissive human fetal brain cells by BK human papovavirus (BKV), the vast majority of the cells were killed by the virus, but rare survivors were recovered after frequent medium changes. These surviving cells grew and formed visible colonies after 5 to 6 weeks and were thereafter established as permanent cell lines. These cells, designated as BK-HFB cells, were persistently infected and shed BKV. Morphologically, they were small polygonal cells and had transformed growth properties. Their plating efficiency on solid substrates or in semisolid medium was high, and they were tumorigenic in athymic nude mice. Cloning experiments in medium containing BKV antiserum revealed that BKV did not persist in the cultures in a simple carrier state. All cloned cell lines were initially T-antigen negative and virus-free. However, every clone began to release BKV and again became persistently infected within 3 weeks after removal of BKV antiserum. After rigorous antibody treatment, four of seven clones still released virus spontaneously upon removal of antiserum; three clones have remained virus-free and are apparently cured. Although these cloned cell lines are T- and V-antigen negative when grown in antiserum-containing medium, they retain "free" or episomal BKV genomes; integrated viral DNA was not detected in any of the clones. These free genomes are indistinguishable from prototype BKV DNA and are found in much larger amounts in virus-shedding cell lines.     

Sesquiterpenes from Porella species

The Porella liverworts contain abundant sesquiterpenes. ent-Biocyclogermacrene, three ent-aromadendrenes, a unique hydrocarbon, α-pinguisene and two drimane type sesquiterpenes were obtained together with the intensly pungent component, tadeonal, from P. vernicosa and P. gracillima. P. macroloba contained the same sesquiterpenes except for the absence of ent-bicyclogermacrene and the ent-aromadendrenes. The fragrant odor of P. perrottetiana was composed of α-pinene and camphor.     

A case of first trimester prenatal diagnosis of 21-hydroxylase deficiency with human complement C4 cDNA probe

Early prenatal diagnosis of 21-hydroxylase (21-OHase) deficiency would enable treatment to be done to protect the fetus from masculinization and/or life-threatening adrenal crisis at birth. We report here the prenatal diagnosis of 21-OHase deficiency with human complement component C4 cDNA to probe DNA from chorionic villi at 10 weeks of gestation. Southern analysis with human C4 cDNA identified TaqI restriction fragment length polymorphisms (RFLPs) in the family. Family analysis with these RELPs showed that the fetus was not affected at greater than 99% probability, because the frequency of recombination between the 21-OHase B gene and the C4 gene would be extremely low.     

Biological and biochemical studies of African green monkey lymphotropic papovavirus.

The growth of African green monkey lymphotropic papovavirus (LPV) in human lymphoblastoid cell line BJA-B was found to be slow and inefficient due to the accumulation of defective particles. An analysis of molecularly cloned LPV DNAs showed that 3 of 19 clones had DNAs that were longer (5.1 kilobases) than the DNAs of the other clones. The 5.1-kilobase DNA was infectious for BJA-B cells, whereas the shorter (4.8-kilobase) molecules were defective. Unlike the wild-type virus, stocks of LPV made from cloned, infectious DNAs were homogeneous and had higher titers. Using stocks of nondefective LPV, we investigated other biological properties. LPV replication in another human B-lymphoblastoid cell line was observed. The virus did not cause tumors when it was inoculated into newborn hamsters. Serological surveys of human and nonhuman primate sera indicated that virtually all primates, including humans, show evidence of infection by viruses antigenically related to LPV.     

Sesquiterpenoids of Riccardia and Pallavicinia species

Riccardia species (Metzgeriales) contain various types of sesquiterpenes. R. jackii produces ent-selinane-, ent-aromadendrane-and ent-bicyclogermacrane-type sesquiterpenes together with (R)-cuparene and α-barbatene. Aneura pinguis (= Riccardia pinguis) is chemically quite different from R. multifida and R. jackii. The former produces a large amount of pinguisone. R. multifida contains 6-(3-methyl-2-butenyl)-indole and (+)-β-elemene as the major components. Pallavicinia longispina (Dilaenaceae; Metzgeriales) produces mainly spathulenol. The chiral properties of the sesquiterpenes isolated from R. jackii are quite similar to those of red algae, Laurencia species.