cis-Diamminedichloroplatinum-mediated crosslinking of nuclear proteins to DNA is cell cycle specific

Immunochemical analysis was employed to investigate the cell cycle-dependent protein-DNA crosslinking by cis-diamminedichloroplatinum II (cis-DDP), in HeLa-S3 cells. Cells synchronized by double thymidine block or hydroxyurea were released into S phase and incubated at 2-h intervals with cis-DDP as they progressed through S1, G2, M, and then into G1 and S phases of the subsequent cycle. Immunoblots of the DNA-crosslinked antigens reacted with antisera to 0.35 M NaCl extract or residue of HeLa S-phase nuclei revealed that several antigens changed their DNA-crosslinking pattern during the progression of HeLa cells through their reproductive cycle.     

Serological studies on Listeria grayi and Listeria murrayi

A cross-agglutination study between somatic antigens from reference strains of Listeria grayi and Listeria murrayi with rabbit antisera was done. L. murrayi antisera reacted, at low titres, with L. grayi but L. grayi antisera did not react with L. murrayi antigen. These results, together with agglutinin-absorption tests, led to the conclusion that the serologic relationship between L. grayi and L. murrayi is not as close as is thought. The two species seem to differ in at least one somatic factor, that might be designated O-XVI for L. grayi and O-XVII for L. murrayi. The serologic relationship of L. grayi and L. murrayi with other serovars of Listeria is discussed. The agglutination titre of 180 healthy ruminants against O-antigens of L. grayi and L. murrayi was also investigated; almost all the sera reacted with the antigens of these species, with similar titres (that reached 640) to those detected against O-antigens of serogroups 1/2 and 4.     

Molecular typing of HLA-B27 alleles

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701—B*2706). These changes are clustered in 1 and 2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoeletric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.     

MAP-1 and MAP-2 binding sites at the C-terminus of beta-tubulin. Studies with synthetic tubulin peptides

The interaction of microtubule-associated proteins MAP-1 and MAP-2 with different peptides containing sequences covering the C-terminal region of beta-tubulin isoforms has been analyzed. Our results indicate that MAP-1 and MAP-2 bind to a common sequence within the variable C-terminal region of the different beta-tubulin isoforms, while MAP-2 also interacts with the subdomain beta (422-434) of the constant region, in agreement with previous results (Maccioni, R.B., Rivas, C., & Vera, J.C. (1988) EMBO J. 7, 1957-1963). The productive interaction of MAP-2 with the latter domain appears to be involved in the assembly of microtubules.     

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha-32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha-32P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.     

Effect of aluminum and lead salts on lipid peroxidation and cell survival in human skin fibroblasts

The aim of this study was to see whether aluminum (Al) and lead (Pb) salts are toxic for cultured human fibroblasts under different experimental conditions, in the controllable situation offered by cell cultures. Cell survival and membrane lipid peroxidation served as markers of Al and Pb toxicity. Evaluation of the living cells was carried out using a colorimetric method, the mitochondrial reduction of 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Lipoperoxidation assay was performed on whole cell homogenates by measuring thiobarbituric acid-reactive substances (TBARS) produced after incubation with ascorbic acid-ferrous sulfate. Al(III) and Pb(II) salts (300 μM) produce a considerable decrease in cell survival after an exposure period of 4 d, evident with the three fetal calf serum concentrations in the culture media: 2, 5, and 10%. Taking into account in vitro cell aging, the cytotoxic effects of Al(III) and Pb(II) are greater in senescent fibroblasts than in young cells. Lead-induced cytotoxicity is higher than Al-induced cytotoxicity. A mechanism that contributes to cellular toxicity is membrane lipid peroxidation; our results demonstrate that Al(III) and Pb(II) ions, 400 μM, exert an antioxidant-like effect or a pro-oxidant action on cell membranes depending on exposure time. We describe significant increases in TBARS formation associated with the presence of 400 μM Al(III) or Pb(II) salts in the culture media. Our study also revealed that these heavy metals induce a cell age-dependent action on membrane lipoperoxidation that is greater in senescent fibroblasts and this could have severe consequences for maintenance of cellular integrity.     

Dmc1 Functions in a Saccharomyces Cerevisiae Meiotic Pathway That Is Largely Independent of the Rad51 Pathway

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.     

Morph-biased male sterility in the tropical distylous shrub Erythroxylum havanense (Erythroxylaceae)

This study explores whether ecological factors, such as pollinators and pollen flow, or variation in pollen and ovule development account for the observed differences (approximately twofold) in the reproductive output of pin and thrum individuals of Erythroxylum havanense. The importance of ecological factors was assessed by means of comparison of the identity of pollinators and the rates of flower visitation, and by performing controlled hand pollinations and measurements of fruit set. In addition, we described the pollen and ovule development of thrum and pin individuals. Our results indicate that pollinators of E. havanense do not distinguish between floral morphs. The differences in fruit set between pin and thrum plants held even after hand pollination and, therefore, the observed differences in reproductive output between floral morphs of E. havanense cannot be explained in terms of asymmetrical pollen flow. There were no differences in the pattern of gynoecium development between the pin and thrum morphs, however androecium development showed marked differences between the morphs, and there was a resemblance between the developmental pathways leading to male sterility of the thrum morph of E. havanense with that of species with cytoplasmic male sterility (CMS).     

Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum

Growth of Corynebacterium glutamicum on mixtures of glucose and fructose leads to simultaneous consumption of both sugars in which the uptake of each sugar is directly related to the expression of the corresponding sugar uptake mechanism. The overall rate of sugar uptake was higher on sugar mixtures than on either glucose or fructose alone and was similar to that observed during sucrose metabolism. The results suggest that sugar uptake limits metabolic rates though, in the case of fructose, overflow metabolism of both lactate and dihydroxyacetone was observed. Such products could reflect a higher flux through glycolysis rather than the pentose pathway during catabolism of fructose. Received: 24 October 1996 / Received revision: 10 January 1997 / Accepted: 10 January 1997     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.