The recovery of normal DNA replication kinetics in ultraviolet-irradiated Chinese hamster cells

The kinetics of DNA replication were analyzed in the second S phase following UV irradiation of Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by treating cells selected in mitosis with hydroxyurea for 9 h. Following UV irradiation, the cells were allowed to progress until the next mitosis; at which time they were resynchronized at the beginning of the second S phase by the same procedure. The kinetics of DNA replication were determined by measuring the proportion of DNA which achieved hybrid buoyant density on CsCl density gradients as a function of the time of incubation in the presence of 5-bromodeoxyuridine.The results of these experiments showed that even though the rate of DNA replication is substantially depressed during the first S phase following UV irradiation with a fluence of 5 J/m², the rate has recovered to the extent that it is indistinguishable from the unirradiated control by the time the cells have entered their second S phase. It was concluded from these observations that the lesions in DNA which caused the rate of DNA replication to be initially depressed during the first S phase have been either removed or modified such that they no longer are able to cause a reduction in the rate of DNA replication in the second S phase following UV irradiation.     

Entropy-driven polymerization of protein from the E66 strain of tobacco mosaic virus

Protein of the tobacco mosaic virus mutant E66 has lysine replacing asparagine of the type strain, vulgare, at position 140. Thus, E66 protein should have one more positive or one less net negative charge than vulgare at pH 6 to 7. To investigate the effect of charge, a comparative study of the polymerization of E66 and vulgare proteins at pH 6.0, 6.2, 6.4, 6.6, and 6.8 at ionic strengths 0.15, 0.10, and 0.05 was made by turbidimetry. Polymerization of E66 protein always proceeded at a lower temperature than vulgare. However, the extent of polymerization was much lower in E66, especially at the higher ionic strengths. Sedimentation velocity results paralleled those from turbidity measurements in that E66 protein polymerizes at lower temperatures than vulgare; the 20 S component is more abundant in E66 protein. Osmotic pressure measurements also show that E66 protein is more polymerized than vulgare, especially at lower pH values. Hydrogen ion titrations of E66 protein were carried out from pH 8 to 5 and back to pH 8 in 0.10 m KCl at three temperatures, 4, 10, and 15 °C. These titrations were reversible when carried out slowly. The isoionic point is near pH 5; thus the charge at pH 7.5 is ?3. The reversible titration results were correlated with the aggregates present at the various pH values and temperatures, determined from the areas under the schlieren peaks in sedimentation velocity experiments. It is found that hydrogen ion binding at the three pH values is correlated with the disappearance of the smallest aggregates and is independent of the type of higher polymer formed. To investigate the effect of ionic strength and pH on the characteristic temperature corresponding to an optical density increment of 0.01 by the method used previously for vulgare, two sets of turbidity measurements were carried out. In the first one the ionic strength was changed from 0.025 to 0.15 in increments of 0.025 at pH 6.0 and 6.4. In the other set, the ionic strength was kept constant at 0.10 and the pH changed from 5.9 to 6.7 in increments of 0.1 pH units. When the analysis of these data was carried out, $\text{Δ}\text{H}{}^1\text{= 30}\text{kcal/mol}$ was obtained. For the salting out constant a value of 1.7 was found, compared to 2.2 for vulgare, a result consistent with the fact that E66 should be less hydrophobic than vulgare. The electrical work term ΔW_el also turns out to be about one-half that for vulgare, which is expected from the lower net negative charge on E66 protein.     

Geographical patterns of persistence in duck guilds

Geographical gradients of persistence in community structure have been suggested to be causally related to underlying gradients of species diversity, environmental variability and/or productivity. In order to test whether the persistence of breeding duck communities was dependent on any one of these three factors, thirty-three years of census data from the Canadian prairie and boreal forest regions was examined along geographical gradients of wetland habitat variability and productivity. For breeding ducks, locally derived patterns of persistence were generally independent of local habitat conditions. Persistence appeared to be related more to patterns of emigration and immigration in response to climatic conditions (i.e., drought) in the southern prairies than to local species richness, wetland habitat variability or productivity. It is suggested, therefore, that analyses of community persistence derived at small spatial scales may be of limited value if the structure of communities is not regulated by local conditions.     

A decade of diabetes: keeping children out of hospital.

OBJECTIVES--To document the number of children aged less than 15 years who developed diabetes and were managed within one large health district, and to evaluate the outcome of those children managed without hospital admission at diagnosis. DESIGN--A retrospective study over 1979-88, when a paediatrician and a physician with special interests in childhood diabetes initiated joint clinics. Data collected from the district diabetes register and files of consultants and health visitors specialising in diabetes. SETTING--Referral of children to consultants in Leicestershire (total population 863,000). MAIN OUTCOME MEASURES--The proportion of children managed without hospital admission, comparison of readmission rates and glycated haemoglobin concentrations between children admitted and those not admitted. RESULTS--Over 10 years 236 children aged 10-14 years developed diabetes (annual incidence rate 12.8/100,000 child population (95% confidence interval 11.3 to 14.7)). In total 138 were not admitted to hospital but received supervised management based at home. Admitted children were younger or acidotic or their family doctors did not contact the diabetes team. Duration of admission declined from seven days in 1979-80 to three days in 1987-8. Ninety two were not admitted to hospital during the 10 years for any reason. Significantly fewer children who received management at home were readmitted for reasons related to diabetes than the group treated in hospital (30 (22%) v 40 (41%); p = 0.004). Concentrations of glycated haemoglobin were no different between the two groups. CONCLUSIONS--Children with newly diagnosed diabetes may be safely and effectively managed out of hospital. Domiciliary or community based management depends on the commitment of consultants specialising in diabetes working in close cooperation with general practitioners, specialist nurses in diabetes, and dietitians.     

A- and B-type lamins are differentially expressed in normal human tissues

 A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies. Accepted: 4 February 1997     

Purification and characterization of chick intestine brush border membrane Effects of 1α(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1α-hydroxyvitamin D₃ treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.