Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding

Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III alpha), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-III beta. The molecular weight of AT-III beta is less than that of AT-III alpha, but physicochemical studies do not indicate measureable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two antithrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-III beta were all 25-30% less than in AT-III alpha. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-III beta inhibited thrombin more rapidly. AT-III beta is also distinguishable from AT-III alpha on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.     

Purification of Bacteroides amylophilus protease

A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-alpha-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.     

Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum.

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.     

Effect of organic loading on nitrification and denitrification in a marine sediment microcosm

Abstract The effects of organic additions on nitrification and dentrification were examined in sediment microcosms. The organic material, heat killed yeast, had a C/N ratio of 7.5 and was added to sieved, homogenized sediments. Four treatments were compared: no addition (control), 30 g dry weight (dw) m⁻² mixed throughout the 10 cm sediment column (30M), 100 g dw m⁻² mixed throughout sediments (100M), and 100 g dw m⁻² mixed into top 1 cm (100S). After the microcosms had been established for 7–11 days, depth of O₂ penetration, sediment-water fluxes and nitrification rates were measured. Nitrification rates were measured using three different techniques: N-serve and acetylene inhibition in intact cores, and nitrification potentials in slurris. Increased organic additions decreased O₂ penetration from 2.7 to 0.2 mm while increasing both O₂ consumption, from 30 to 70 mmol O₂ m⁻² d⁻¹, and NO₃⁻ flux into sediments. Nitrification rates in intact cores were similar for the two methods. Highest rates occurred in the 30M treatment, while the lowest rate was measured in the 100S treatment. Total denitrification rates (estimated from nitrification and nitrate fluxes) increased with increased organic addition, because of the high concentrations of NO₃⁻ (40 μM) in the overlaying water. The ratio of nitrification: denitrification was used as an indication of the importance of nitrification as the NO₃⁻ supply for denitrificaion. This ratio decreased from 1.55 to 0.05 iwth increase organic addition.     

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non- transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8- month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF- II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I     

Apolipoprotein A-I decreases neutrophil degranulation and superoxide production.

Neutrophils participate in the acute phase response and are often associated with tissue injury in a number of inflammatory disorders. The acute phase response is accompanied by alterations in the metabolism of apolipoprotein A-I and high density lipoprotein (HDL). Structural considerations led to studies investigating the effect of purified HDL and apolipoprotein A-I on neutrophil degranulation and superoxide production. Apolipoprotein A-I but not HDL inhibited IgG-induced neutrophil activation by about 60% as measured by degranulation and superoxide production. This suggests that the lipid-associating amphipathic helical domains of apolipoprotein A-I mediate this effect. In support of this was finding inhibitory effects with two synthetic model lipid-associating amphipathic helix peptide analogs. Apolipoprotein A-I, containing tandem repeating amphipathic helical domains, was approximately ten times more effective than the two peptide analogs and inhibited neutrophil activation at well below physiologic concentrations. Competitive binding studies indicate that resting neutrophils have approximately 190,000 (Kd = 1.7 x 10(-7)) binding sites per cell for apolipoprotein A-I, consistent with a ligand-receptor interaction. These observations suggest that apolipoprotein A-I may play an important role in regulating neutrophil function during the inflammatory response.     

Tel-1 Transposon-like Elements of Tetrahymena Thermophila Are Associated with Micronuclear Genome Rearrangements

The micronuclear genome of Tetrahymena thermophila contains Tel-1 elements that structurally resemble transposons. Here we present molecular evidence that Tel-1 transposon-like elements are mobile. The arrangements of Tel-1 elements in the micronuclear genomes of several T. thermophila strains and cell lines were assayed by Southern blotting. The molecular evidence for Tel-1 transposition is most striking in strains that have undergone unusual laboratory-induced meioses. The genetic history of the strains exhibiting evidence of Tel-1 transposition is consistent with periods of genome restructuring in response to genomic ``shock' that B. McClintock has suggested could result in transposon activation.     

Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway.

We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.