Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage lambda. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type. In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA. The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant. Binding was increased specifically to DNA rather than to nucleic acids in general. The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells. The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Yearly maps of summer temperatures in Western Europe from A.D. 1750 to 1975 and Western North America from 1600 to 1982:

The primary purpose of this publication is to make available two series of historical maps showing summer (April–September mean) temperatures over western Europe from A.D. 1750 and western North America from A.D. 1600. These maps have been reconstructed using networks of temperature-sensitive tree-ring chronologies as part of a continuing collaboration between the Swiss Federal Institute for Forest, Snow and Landscape Research (FSL) at Birmensdorf, Switzerland and the Climatic Research Unit (CRU) at the University of East Anglia, Norwich, U.K. The maps were reconstructed using relatively long maximum latewood density chronologies selected from the extensive networks of tree-ring data produced at the FSL. The reconstructions were produced using a spatial regression technique developed at CRU.The basis for the reconstructions are wood samples from various conifers growing at cool-moist sites close to the alpine or boreal timberlines. This material was analysed by x-ray densitometry and the resulting maximum latewood density chronologies were calibrated against meteorological data over a 100-year calibration period.These results emphasize the value of dendroclimatological research which can provide continentalscale reconstructions of past climate which represent a potential resource for climatologists, biologists, and historians with an interest in the spatial variability of summer temperatures over the middle to high latitudes.     

Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Mortality differentials within large American cities in 1890

This article presents a multiple regression analysis of demographic and social data for 335 wards in 17 American cities in 1890. The most important findings are: (1) Density effects on mortality were uniformly positive and statistically significant; the magnitude of these effects was much greater for child mortality than for adult mortality; and child mortality was more sensitive to persons per dwelling than to persons per acre. (2) Unsanitary conditions, as measured by the city-specific typhoid fever death rate, significantly increased mortality and child mortality was much more sensitive in this respect than adult mortality. (3) Given the same age composition and population density, foreign-born whites, native-born whites, and the colored population had about the same adult death rate.     

Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.