Unique acceptors for poly(ADP-ribose) in resting,proliferating and DNA-damaged human lymphocytes

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.     

Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing

Human lymphocytes stored in the frozen state may be thawed, placed on cytotoxicity plates, refrozen, rethawed and used for screening sera or tissue-typing of the cells. The simple procedure described uses only a ?90 °C refrigerator for both freezing and storage of the cells. The technique permits a laboratory to collect a variety of cells over a long period, so that a set of test plates with cells from 10 to 20 donors can be prepared when a convenient number of donor cells are available. Also, the refrozen cells in cytotoxicity test plates may be warmed to the temperature of dry ice for 24 hr, returned to the refrigerator set at a slightly lower temperature, and at a later time, these cells may be thawed and used for serum screening. In view of these results, it appears possible to ship the refrozen cells from one laboratory to another using simple dry ice storage during the transfer. Negative reactions due to soluble antigens in the suspending sera can be obviated by washing out these sera and replacing them with medium 199 or alternatively, fetal calf serum can be used to replace the human serum in the suspending media.     

Stationary underwater prey missed by reef herons,Egretta gularis: head position and light refraction at the moment of strike

Summary This paper attempts to verify the importance of spatial positioning of the eyes of reef heronsEgretta gularis schistacea, when coping with light refraction at the air-water interface. The herons' striking of prey, while their approach angle was restricted, was observed. (a) The herons' capture success in the restricted situation was markedly lower than in the unrestricted situation. (b) The points of strike (STR) in unsuccessful strikes differed from those of successful strikes, and from those of the unrestricted situation. (c) The larger the difference between the observed and the predicted ratio of prey depth to apparent prey depth, the higher the probability of missing a prey. These results support predictions of a model presented elsewhere (Katzir and Intrator 1987) that a heron will attempt to reach spatial positions at which prey's real depth and apparent depth are linearly correlated.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages

Generation of nitric oxide (NO.), an autacoid with vasorelaxant and cytotoxic properties, requires at least three cytosolic components in mouse macrophages besides L-arginine and NADPH. One or more components appear after induction by immunologic stimuli; two or more are present in both activated and non-activated macrophages. The constitutive factors can be separated on a Mr approximately 30,000 cut-off filter into high Mr fraction (HF) and low Mr fraction (LF) (Stuehr, D. J., Kwon, N. S., Gross, S. S., Thiel, B. A., Levi, R., and Nathan, C. F. (1989) Biochem. Biophys. Res. Commun. 161, 420-426). Herein we characterize the major active component in LF. The active component was dialyzable (Mr less than approximately 1,000), water soluble, and cationic at acidic to neutral pH. Fractionation on a C18 column in an acetonitrile/water gradient yielded one broad peak of activity, most of which corresponded to a fluorophore with the excitation/emission spectra of biopterins. Gas chromatography isolated a species in this peak with the mass spectrum of biopterin. Of 14 pteridines tested, only 7,8-dihydrobiopterin (H2biopterin) or 5,6,7,8-tetrahydrobiopterin (H4biopterin) could replace LF in synergizing with HF and the inducible component(s) to generate NO-2 and NO-3, the accumulating oxidation products of NO.. Half-maximal activity required 20-30 nM reduced biopterins. LFs from three cell lines were active in proportion to their content of biopterins; addition of reduced biopterins restored activity to LF from biopterin-deficient cells. Enhancement of NO-2 generation in the presence of H2biopterin but not H4biopterin was abolished by methotrexate and aminopterin, inhibitors of dihydrofolate reductase. These findings implicate a redox cycle in which the generation of NO. is facilitated by catalytic amounts of H4biopterin.     

Evidence for the existence of RNA of Ca(2+)-channel alpha 2/delta subunit in Xenopus oocytes.

Ba(2+)-currents (IBa) through voltage-dependent Ca(2+)-channels were studied in Xenopus oocytes injected with RNA from several excitable tissues, using the two-electrode voltage-clamp technique. Previous studies have shown that the expression of cardiac Ca(2+)-channels can be suppressed by an hybrid-arrest procedure that includes co-injection of the tissue-derived RNA with an 'antisense' oligonucleotide complementary to a part of RNA coding for the Ca(2+)-channel alpha 1 subunit. In this study, this method was used to investigate the role of the alpha 2/delta subunit. Co-injection of RNA extracted from either rabbit heart, rat brain or rat skeletal muscle (SkM) with 'antisense' oligonucleotides complementary to the alpha 2/delta subunit RNA did not substantially affect the expression of IBa in the oocytes. Using the Northern blot hybridization method, it was shown that native oocytes contain large amounts of alpha 2/delta subunit RNA of Ca(2+)-channel. It is proposed that te oligonucleotide treatment fails to eliminate the alpha 2/delta RNA because of the vast excess of endogenous alpha 2/delta RNA. These results impose a limit on the use of the hybrid-arrest method.     

Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.     

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.     

Biogeographic context is related to local scale tree demography,co-occurrence and functional differentiation

Identifying the drivers of community structure and dynamics is a major pursuit in ecology. Emphasis is typically placed on the importance of local scale interactions when attempting to explain these fundamental ecological patterns. However, regional scale phenomena are also important predictors. The importance of regional scale context should be more evident in assemblages where multiple species are close to their range margins. Here, we test the importance of regional scale context using data from a temperate forest plot that contains two species groups – one near its northern range limit and one near its southern range limit. We show the proximity of species to their southern or northern range margins is linked to local scale co-occurrence, similarity in gene expression responses to a key environmental driver, demographic performance and inter-specific variation in conspecific negative density dependence. In sum, many of the key local scale patterns and processes of interest to community ecologists are linked to biogeographic context that is frequently ignored.