Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation.

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Properties of R plasmid R772 and the corresponding pilus-specific phage PR772.

R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 x 10(6). It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1.26 g cm(-3). These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.     

Membrane compartmentalized ATP and its preferential use by the Na, K-ATPase of human red cell ghosts

This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na, K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [γ-(32)P]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37 degrees C, but not at 0 degrees C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [gamma-(32)P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as (32)P(i) liberated during the subsequent incubation at 0 degrees C, reflects completely the differential effects of preincubation with ATP on (32)P incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na, K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na, K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.     

Generation of active TGF-beta by prostatic cell cocultures using novel basal and luminal prostatic epithelial cell lines

Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays.     

Structure and synthesis of ether-linked proteracacinidin and promelacacinidin proanthocyanidins from Acacia caffra

Two new ether-linked proanthocyanidins, epioritin-(4beta-->3)-epioritin-4beta-ol and epimesquitol-(4beta-->4)-epioritin-4beta-ol, were isolated from the heartwood of Acacia caffra. Their structures and absolute configurations were established by spectroscopic methods and syntheses.     

Repellent effects on Anopheles arabiensis biting humans in Kruger Park, South Africa

Abstract. Distribution of biting sites on the human body by the malaria vector Anopheles arabiensis Patton (Diptera: Culicidae) was investigated near a source of mosquitoes in the Kruger National Park, South Africa. Eight adult male volunteers (2 teams × 2 pairs of subjects) conducted human bait collections while seated on camp chairs in the open-air, wearing only short trousers (no shirt, socks or shoes). Mosquito collections during 18.30–22.30 hours on five consecutive nights in April 1998 yielded a total of 679 An. arabiensis females biting subjects with or without their ankles and feet treated with deet insect repellent (15% diethyl-3-methylbenzamide, Tabard™ lotion). On subjects whose feet and ankles were smeared with repellent, 160 An. arabiensis females were captured biting in 60 man-hours: 88.1% on the legs, 1.4% on the arms and 1.2% on other parts of the body, but none on the repellent-treated feet or ankles. On subjects without repellent treatment, 519 An. arabiensis were caught biting in 60 man-hours: 81.1% on feet and ankles, 16.4% on legs, 1.4% on arms and 1.2% on the rest of the body. For individual subjects, the reduction of An. arabiensis bites ranged from 36.4 to 78.2% (mean protection 69.2%). Results of this study confirm previous findings that, in this part of South Africa − inhabited only by wildlife − when people sit outside during the evening An. arabiensis prefers to bite their lower limbs: 97.5% below the knees. Overall, the number of bites by the malaria vector An. arabiensis was reduced more than three-fold (from 26 to 8/person/evening), simply by treating ankles and feet with a consumer brand of deet repellent. Whether or not this provides a satisfactory degree of protection against malaria risk would depend on the malaria sporozoite rate in the malaria vector population.     

Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters

Deficiency of -sarcoglycan(-SG), a component of the dystrophin-glycoprotein complex, causescardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Usingcultured myotubes prepared from skeletal muscle of normal and Bio14.6hamsters (J2N-k strain), we investigated the possibility that the-SG deficiency may lead to alterations in ionic conductances, whichmay ultimately lead to myocyte damage. In cell-attached patches (withBa²⁺ as the charge carrier), an ~20-pS channel wasobserved in both control and Bio14.6 myotubes. This channel is alsopermeable to K⁺ and Na⁺ but not toCl. Channel activity was increased by pressure-inducedstretch and was reduced by GdCl₃ (>5 µM). The basal openprobability of this channel was fourfold higher in Bio14.6 myotubes,with longer open and shorter closed times. This was mimicked bydepolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes,the unidirectional basal Ca²⁺ influx was enhanced comparedwith control. This Ca²⁺ influx was sensitive toGdCl₃, signifying that stretch-activated cation channelsmay have been responsible for Ca²⁺ influx in Bio14.6hamster myotubes. These results suggest a possible mechanism by whichcell damage might occur in this animal model of muscular dystrophy.

     

Bioassay and biochemical analyses of insecticide resistance in southern African Anopheles funestus (Diptera: Culicidae)

Anopheles funestus Giles has been implicated as a major malaria vector in sub-Saharan Africa where pyrethroid insecticides are widely used in agriculture and public health. Samples of this species from northern Kwazulu/Natal in South Africa and the Beluluane region of southern Mozambique showed evidence of resistance to pyrethroid insecticides. Insecticide exposure, synergist and biochemical assays conducted on A. funestus suggested that elevated levels of mixed function oxidases were responsible for the detoxification of pyrethroids in resistant mosquitoes in these areas. The data suggested that this mechanism was also conferring cross-resistance to the carbamate insecticide propoxur.