The frequency of the ΔF508 mutation on cystic fibrosis chromosomes in Israeli families: correlation to CF haplotypes in Jewish communities and Arabs

Summary We have analysed the distribution of the ΔF508 mutation and the haplotypes of cystic fibrosis (CF) bearing chromosomes among the Israeli CF population. The population was classified according to its ethnic origin and included 3 groups, Ashkenazi Jews, Sephardic/Oriental Jews and Arabs. Haplotype B (KM19 allele 2, XV2c allele 1) was found to be the predominant haplotype in all groups but in each of them the haplotype distribution was different. The ΔF508 mutation was present in all groups and accounts for 32% of the CF mutations. It was mainly associated with the B haplotype but only one third of the CF chromosomes with this haplotype carry the ΔF508 mutation. This work is dedicated to Dr. Ruth Voss who initiated the CF study in Israel and was tragically killed in a car accident on 7 August 1988     

Effect of Metal-Rich Sewage Sludge Application on the Bacterial Communities of Grasslands

The effect of long-term application of heavy metal-laden sewage sludge on the total heterotrophic aerobic and the cadmium-resistant soil bacterial communities was studied. Gram-positive bacteria were completely absent from resistant communities. These findings suggest that this group is highly susceptible to Cd. Shannon's diversity indices estimated for total communities did not reveal negative effects on the communities that developed in the presence of sludge. However, Cd-resistant communities isolated from long-term sludge-amended soils were more diverse than the resistant communities from a control sample, suggesting that adaptation to Cd as a stressor had occurred in the presence of sludge constituents. This higher diversity was attributed to Cd resistance in pseudomonads and gram-negative fermenters. Resistance did not develop by dissemination of Cd resistance plasmids, because these were rarely detected in the genomes of resistant strains.     

Hyperscums and the population dynamics of Microcystis aeruginosa

Conceptual models, based on 7 years of data, are constructedto simulate the annual cycle and population dynamics of Microcystisaeruginosa in hypertrophic, warm monomictic Hartbees-poort Dam,South Africa in order to assess the role of hyperscum formation.In Hartbeespoort Dam the large summer planktonic population(mean epilimnion biovolumes of 20–50 mm³ I^–1) andthe low wind speed resulted in the formation of hyperscums (thick,crusted accumulations of floating cyanobacteria at wind-protectedsites) containing up to 50% of the total standing crop for 2–3months in 4 out of 5 years. In years in which hyperscums formedthe post-maximal summer population maintained itself throughoutautumn and into late winter before declining to the annual nadir(>1000 cells ml^–1). When hyperscums did not form, orwere artificially removed, the population fell to similarlylow levels as early as May (autumn) and remained small untilthe spring growth phase began. Microcystis cells decompose inthe upper layers of a hyperscum, but this is not a major lossto the planktonic population. Hyperscums are refuges which helpmaintain large planktonic standing crops during winter whengrowth is not possible but have no effect on the long-term (perennial)survival of Microcystis.     

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.     

Microtubule inhibitors differentially affect translational movement,cell surface expression,and endocytosis of transferrin receptors in K562 cells

We used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein-conjugated transferrin (FITC-Tf). Coordinated decreases in surface fluorescence counts, the photobleachig parameter K, and transferrin receptor fractional mobility were observed as FITC-Tf was cleared from the cell surface by receptor-mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC-Tf uptake at 37°C and 21°C were calculated to be 0.10-0.15 min^?1 and 0.02–0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by β-lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtuble inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule-stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug-resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor-mediated transferrin uptake in K562 cells. © 1994 Wiley-Liss, Inc.     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.     

PHOTOADAPTATION AND THE “PACKAGE” EFFECT IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

In the marine unicellular chlorophyte, Dunaliella tertiolecta Butcher, the spectrally averaged m vivo absorption cross section, normalized to chlorophyll a (so-called a* values), vary two-fold in response to changes in growth irradiance. We used a kinetic approach to examine the specific factors which account for these changes in optical properties as cells photoadapt. Using Triton X-100 to solubilize membranes, we were able to differentiate between “package” effects and pigmentation effects. Our analyses suggest that 43–49% of the variability in a* is due to changes in pigmentation, whereas 51–57% is due to the “package” effect. Further analyses revealed that changes in cell sue did not significantly affect packaging, while thylakoid stacking and the transparency of thylakoid membranes were important factors. Our results suggest that thylakoid membrane protein/lipid ratios change during photoadaptation, and these changes influence the effective rate of light harvesting per unit chlorophyll a.