Inactivation of p16 in Human Mammary Epithelial Cells by CpG Island Methylation

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G₁ termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16^INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.     

Effects of prior self-pollination on outcrossed seed set in tristylous Pontederia sagittata (Pontederiaceae)

The potential inhibitory effects of incompatible pollen on outcrossed seed set were investigated in mass-flowering, self-incompatible, tristylous Pontederia sagittata. Prior application of self pollen, followed after 2, 4, or 6 h by compatible pollen, was conducted on five genotypes of each of the three style morphs under uniform glasshouse conditions. The greatest reductions in seed set occurred in pollinations of the long-styled (L) morph at the 6 h time interval. Smaller reductions were also found for this treatment in the mid-styled (M) morph. No significant reductions in seed set were observed in the short-styled (S) morph or in the other morphs at shorter time intervals. Observations of pollen germination and pollen tube growth indicated that the lack of inhibitory effects in the S morph may occur because relatively few pollen grains adhered to stigmas in selfpollinations. In the L and M morphs, early germination of self pollen may cause physical clogging of the stigma and style, resulting in a reduced number of compatible pollen tubes in styles. Observations of the structural integrity of styles indicated that prior germination of self pollen resulted in more rapid onset of pistil senescence, particularly in the L morph. These influences may contribute to the morph-specific differences in seed set observed following prior self-pollination of outcrossed flowers. The negative effects of incompatible pollen are likely to be most evident where ecological factors cause delays in the delivery of outcross pollen to stigmas.     

Absorption of non-haem iron from food during normal pregnancy.

OBJECTIVE--To determine whether the increased iron demands of pregnancy could be met by increased absorption from dietary sources. DESIGN AND SETTING--Longitudinal prospective study in the research unit of a maternity hospital. SUBJECTS--12 normal pregnant women. INTERVENTIONS--At 12, 24, and 36 weeks'' gestation (within one week) and 16-24 weeks after delivery women ate a breakfast of meat, bread, and orange juice (3.2 mg iron), extrinsically labelled with the stable isotope iron-54 (2.8 mg); the stable isotope iron-57 (200 micrograms) was given intravenously. MAIN OUTCOME MEASURES--Serum samples were taken for 10 hours after administration of the isotopes; ratios of the isotopes were measured by inductively coupled plasma mass spectrometry, and the absorption of oral iron was calculated. RESULTS--The geometric mean (95% confidence interval) absorption of iron at 12, 24, and 36 weeks'' gestation was 7% (5% to 11%), 36% (28% to 47%), and 66% (57% to 76%) respectively. At 16-24 weeks after delivery the absorption was 11% (6% to 21%). The mean increase in absorption at 36 weeks (compared with that at 12 weeks) was 9.1 times (6.0 to 13.7). One pregnant woman developed iron deficiency anaemia but was otherwise indistinguishable from the others. CONCLUSIONS--An increase in the absorption of iron from food is a physiological consequence of normal pregnancy, not the result of developing anaemia during pregnancy, and such an increase is large enough to meet the increased requirements of pregnancy provided that the dietary intake is adequate.     

Localization of carbohydrate and DNA in cyst-like structures from a human intestinal spirochaete

This study examined the cyst-like structures found in human intestinal spirochaetes by transmission electron microscopy and by histochemical and immunocytochemical analysis. A human intestinal spirochaete which morphologically resembled other intestinal spirochaetes was grown anaerobically on blood agar plates and in Tryptone Soya broth (Oxoid) and harvested by centrifugation after 8 days growth. Specimens were either conventionally fixed for transmission electron microscopy or fixed in 0.5% glutaraldehyde in 0.1M sodium cacodylate buffer and embedded in LR White resin for immunocytochemistry. En bloc histochemical investigation using a periodic acid-thiosemicarbazide-silver proteinate technique was undertaken for the localization of carbohydrate. A post-embedding immunogold labelling technique was used on ultrathin sections to label DNA. Results from the histochemical study demonstrated a reaction product which was confined to the cytoplasm of mature spirochaetes and in the central bodies within the cysts. Immunogold labelling demonstrated the presence of DNA in both the mature protoplasmic cylinders and in the central bodies. The results of the present study indicate that spirochaetal cysts are highly organized structures, which contain both DNA and carbohydrate. These findings are compatible with the view that these structures have a functional role rather than representing degenerative artifacts.     

A comparison of the fate and effects of prochloraz in artifical and natural sediments

The current draft EC regulatory guidance document for the authorisation of plant protection products, directive EEC 91/414, contains provision for testing the potential toxicity of pesticides to sediment dwelling organisms. Development of an appropriate method is currently in progress, by a German regulatory and industry working group. The compounds which are expected to trigger sediment toxicity data are likely to be those which are relatively persistent, with a high K _ow, with the potential for significant adsorption to sediments, and may have previously demonstrated toxicity to aquatic organisms. The current proposal from this working group involves an artificial sediment, prepared in accordance with OECD guideline 207, as used for earthworm toxicity tests. The implications of using this sediment on the distribution, fate and availability of the compound under test are considered. This paper presents toxicity data for Chironomus riparius generated using radiolabelled Prochloraz^® in systems using this artificial sediment and two natural sediments. The fate of prochloraz was also investigated in the same systems and compared with data generated from sediment/water studies carried out in accordance with regulatory guidelines. At the maximum Predicted Environmental Concentration (PEC) of 195 g^-1 nominal, prochloraz had no effects on the survival and development of Chironomus riparius. The fate of the compound in the systems used was enhanced possibly as a result of photodegradation.     

An evaluation of antibiotics for the elimination ofXanthomonas campestris pv.pelargonii (Brown) fromPelargonium x domesticum cv. ‘Grand Slam’ explants in vitro

A range of antibiotics was evaluated for activity againstXanthomonas campestris pv.pelargonii (Xep) on diagnostic sensitivity testing and plant tissue culture media. Many of the antibiotics showed reduced or no activity on the latter. Tetracycline and cefotaxime, chosen for further investigation, were screened for light stability under plant culture regimes. Tetracycline was inactivated in photosynthetic photon fluxes of 22 mol m^-2 s^-1 and above. The minimum bacteriocidal concentration of cefotaxime was determined in bacteriological and plant tissue culture media. Cefotaxime was further tested for phytoxicity and ability to eliminate Xcp from deliberately infected explants. Cefotaxime was shown to eliminate contamination and stimulate the growth of the plant tissue cultures up 500 mg l^-1.Abbreviations DST diagnostic sensitivity testing medium - MBC minimum bacteriocidal concentration - TCM half-strength Murashige & Skoog (1962) basal plant tissue culture medium - Xcp Xanthomonas campestris pv.pelargonii - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - OD optical density - PPF photosynthetic photon flux     

Lysosomal acid proteinase of rabbit liver

1. The interference mechanism of carbonyl cyanide m-chlorophenylhydrazone with the respiratory process and with phosphorylation coupled to respiration has been investigated in resting cells of Escherichia coli. 2. Preincubation of the cells with carbonyl cyanide m-chlorophenylhydrazone in the absence of substrate caused strong inhibition of succinate oxidation. The inactivation of the respiratory system proved to be time-dependent and temperature-dependent and could be arrested by adding the substrate. Inhibition of incorporation of ³²P into acid-soluble organic phosphate esters exceeded the inhibition of oxygen uptake. 3. In contrast with succinate, the rate of oxidation of glucose was increased by carbonyl cyanide m-chlorophenylhydrazone. The sensitivity of other substrates to the inhibitor was less than that of succinate. 4. Various observations are described in support of the view that respiratory inhibition induced by carbonyl cyanide m-chlorophenylhydrazone is a result of its interference with ATP synthesis. The capacity of a given substrate to increase intracellular ATP concentration appeared to be directly related to its resistance to inhibition. In cell-free extracts carbonyl cyanide m-chlorophenylhydrazone still suppressed ³²P incorporation but had no effect on respiration. 5. Carbonyl cyanide m-chlorophenylhydrazone-induced stimulation of glucose oxidation and the acceleration of succinate oxidation by ADP or AMP in cells rendered permeable to nucleotides are tentatively interpreted as an indication that a certain part of respiration in E. coli is under phosphate-acceptor-mediated control.