In vitro senescence of human keratinocyte cultures

Human keratinocytes have been serially cultivated in low (0.015 mM) and high (1.8 mM) calcium containing medium. The calcium concentration of the growth medium significantly influenced the cell growth period in vitro. Cells grown in low calcium medium underwent 35-40 population doublings over 16-17 passages, while cells grown in high calcium medium ceased to proliferate after 20 population doublings over 7 passages. Changing the keratinocytes from one in vitro environment to the other drastically altered the lifespan in culture of populations derived from the same primary tissue. The degree of DNA methylation of human keratinocytes was shown to decrease with age in both high and low calcium culture conditions but does not appear to be associated with differentiation.     

Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.     

Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of a hydrophobic peptide in membrane bilayers by solid-state nuclear magnetic resonance

Solid-state NMR studies of the dynamics of a synthetic hydrophobic peptide, tert-butyloxycarbonylleucylphenylalanine methyl ester (Boc-Leu-Phe-OMe), in phospholipid bilayers are described. Motionally averaged powder pattern line shapes from 2H- and 15N-labeled backbone and side-chain sites of the peptide in phospholipid bilayers demonstrate the presence of both overall and internal reorientations of substantial amplitude. The peptide motions are shown to be strongly influenced by the motions of the lipids.     

Radiosensitization, pharmacokinetics, and toxicity of a 2-nitroimidazole nucleoside (RA-263)

A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties. The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve. Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells. The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole. The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole. The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation. The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves. The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer. Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively. The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS. Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests. This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli.     

Conformational comparison of cyclic peptide and pseudopeptide structures with intramolecular hydrogen bonding

An nmr spectral comparison of a model cyclic pentapeptide cyclo(Gly-Pro-Gly-D-Phe-Pro) with an analogous pseudopeptide has been made. The pseudopeptide contains a ψ[CH₂S] amide bond replacement at the only amide linkage that, in the model, is not involved in an intramolecular hydrogen bond. Both proton and carbon-13 nmr spectral evidence confirms the retention of β- and γ-turns in the pseudopeptide in chloroform. Characteristic chemical shifts, temperature dependence, and glycine α-resonances support this interpretation. However, evidence of a more flexible conformation involving cis–trans proline isomerism is seen on addition of dimethylsulfoxide.     

Studies of sibling Drosophila species from Laguna Verde, Veracruz, Mexico: I. Species frequencies, viability, desiccation resistance, and vagility

The sibling species Drosophila melanogaster and D. simulans were collected at Laguna Verde, Veracruz, Mexico. D. melanogaster was found in significantly greater frequency than was D. simulans. Ten isofemale lines of each species were tested for egg to adult viability, desiccation resistance, and vagility. D. melanogaster surpassed D. simulans in all three characteristics. The findings are discussed with reference to the climatic conditions at Laguna Verde and the expected effect of such an environment on the relative frequencies of these species. The dichotomous results in regard to desiccation resistance and vagility that were observed between recently collected D. melanogaster and the Oregon-R laboratory stock of that species are also discussed.     

Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex

The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types. Dot plots indicate that these types were not related by conspicuous rearrangements or inversions. More than one ITS type was often found in the same taxon, and two of three ITS types span species boundaries, indicating their presence prior to speciation. These ITS sequences showed essentially no positional homology with the nearest sequenced relative, tomato. In contrast, the 5.8S region was relatively unvaried, with 8 of 162 sites varied in the sample among all eight taxa. The phylogeny inferred by the most common ITS sequence type, rooted by the two other ITS types, agreed with isozymes in showing the distinctness of M. nudatus, M. laciniatus, and M. tilingii from the other five taxa.      

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Assortative mating and gene flow in the Lesser Snow Goose: A modelling approach

Nonrandom mating with respect to color is well documented in the dimorphic Lesser Snow Goose. A set of mathematical models and data from the long-term study at La Pérouse Bay, Manitoba, Canada are used to examine various hypotheses advanced to explain the mechanisms behind the assortative mating. Among the factors considered are mate choice based on familial color, accidental formation of genetically unrelated families, and nonuniform distribution of colors in the region where mate selection occurs.