Human renal clear cell carcinoma: Establishment and characterization of a new cell line (G-2101)

Summary A human cell line has been established from a renal adenocarcinoma rib metastasis of a 58-y-old male. This cell line has been maintained in continuous culture for 20 mo. through more than 50 passages. It displays simulataneous expression of the intermediate filaments cytokeratin and vimentin. Flow cytometric analysis of DNA content reveals a major hyperdiploid population. This work was supported in part by a grant from Triton Biosciences, Inc.     

Immunologic comparisons of Pneumocystis carinii strains obtained from rats, ferrets, and mice using convalescent sera from the same sources.

Pneumocystis carinii (Pc) infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or trans-tracheal inoculation of Pc obtained from infected lungs of the homologous species (rat, mouse). Convalescent antisera were obtained by stopping dexamethasone treatment after 2-4 wk and allowing 5-8 wk for recovery. Parasites from infected lungs were purified by differential filtration, solubilized in loading buffer, subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and blotted to polyvinylidene fluoride sheets for Western analysis. Antisera from each animal species were reacted on Western blots of antigens from rat, ferret, and mouse. Each combination of antigen and antibody from the same species of animal showed reaction with 5 or more bands of Pc antigen. Convalescent mouse antibody did not react with rat or ferret antigens. Convalescent rat antibody reacted with a mouse antigen at about 66 kDa but not with ferret antigen, and convalescent ferret antibody showed minimal, probably non-specific reactions with both rat and mouse antigens. Variations in reactions indicate antigenic differences in Pc strains infecting these animals.     

A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.     

Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

Effects of in vitro growth phase on the pathogenesis of Salmonella typhimurium in mice

The growth phase of a bacterial (Salmonella typhimurium) culture was shown to have pronounced effects on the pathogenic properties of the harvested bacteria. Salmonellae obtained from a culture in primary (exponential) growth phase (PP) were more readily cleared from the blood and more readily killed by phagocytes than were salmonellae obtained from a more slowly growing secondary growth phase (SP) culture. PP salmonellae were observed to cause death of mice sooner than SP salmonellae. This appeared to be because the more rapid growth of PP, as compared to SP, salmonellae continued in the liver and spleen for several hours following intravenous injection, and more than compensated for their high in vivo death rate. As a result, within 4 h there were approximately 10-fold more live salmonellae in the spleens and livers of mice that had received PP, as compared to SP, salmonellae. This 10-fold difference was maintained until the death of the mice, indicating that after the first 4 h post-inoculation, the net in vivo growth of the salmonellae was the same regardless of their growth phase in the inoculating culture. This transition between PP and SP salmonellae occurred long before a dense stationary phase culture was obtained. Salmonellae grown in minimal media exhibited the biological properties of SP salmonellae and never entered as rapid a growth phase as did salmonellae in complete media.     

Recombinant human interleukin 1 alpha: purification and biological characterization

Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1. By using the murine IL 1 cDNA as a probe, we have isolated its human homolog from cDNA generated to lipopolysaccharide-stimulated human leukocyte mRNA. Nucleotide sequence analysis of this cDNA predicts a protein of analysis of this cDNA predicts a protein of 271 amino acids (termed IL 1 alpha) which shows congruent to 61% homology to its murine counterpart but only 27% homology to a recently characterized human IL 1 precursor (IL 1 beta). We have expressed the carboxy-terminal 154 amino acids of IL 1 alpha in E. coli, purified this protein to homogeneity, and have compared it with pure recombinant murine IL 1 in several different IL 1 assays based on murine and human cells. Recombinant IL 1 is capable of stimulating T cell and fibroblast proliferation and inducing fibroblast collagenase and prostaglandin production, thus proving that a single molecule has many of the activities previously ascribed to only partially purified IL 1 preparations. Our results indicate that there exists a family of at least two human IL 1 genes (alpha and beta) whose dissimilar protein products have similar biological activities.     

Metabolic adaptations in goat mammary tissue during pregnancy and lactation

Metabolic adaptations of goat mammary tissue during pregnancy and lactation were monitored in serial biopsies of the tissue. Changes in the synthetic capacity of secretory cells were studied by combining measurements of enzyme activities with short-term culture of mammary explants to measure lactose, casein and total protein synthesis. By these criteria, the main phase of mammary differentiation began in late pregnancy and was essentially complete by Week 5 of lactation, coinciding with the achievement of peak milk yield. While milk yield declined after Week 5, the activities of key enzymes expressed per mg DNA and the rates of lactose and casein synthesis in mammary explants were maintained over a considerable period. The results suggest that changes in the synthetic capacity of epithelial cells may account for much of the rise in milk yield in early lactation, but are not responsible for the declining phase of milk production characteristic of lactation in ruminants.