Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.     

Feline leukemia virus: biochemical and immunological characterization of gag gene-coded structural proteins.

The major non-glycosylated structural proteins of feline leukemia virus have been isolated, and competition immunoassays have been developed for each. These proteins include the 27,000- to 30,000-molecular-weight major internal antigen designated p30, a 15,000-molecular-weight protein (p15), an acidic protein of 12,000 molecular weight (p12), and a highly basic 10,000-molecular-weight protein (p10). Immunologically and biochemically corresponding proteins of feline and murine leukemia viruses have been identified. and, on the basis of analogy to the known sequence of a prototype type C virus of mouse origin, the map order of the gag region of the feline type C viral genome has been tentatively deduced as NH2-p15-p12-p10-COOH. The demonstration of two feline leukemia virus gag gene-coded proteins, p15 and p12, expressed in the form of an uncleaved precursor in a mink cell line nonproductively transformed by feline sarcoma virus provides indirect support for the proposed sequence.     

Structural studies of the haemophilus influenzae type f capsular polysaccharide

The structure of the Haemophilus influenzae type f capsular polysaccharide was studied by chemical and nuclear magnetic resonance spectroscopic techniques. The repeating unit of the polysaccharide was found to be

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Translation of type C viral RNAs in Xenopus laevis oocytes: evidence that the 120,000-molecular-weight polyprotein expressed in Abelson leukemia virus-transformed cells is virus coded.

The genomic RNA of Abelson leukemia virus (AbLV) has been purified and translated in Xenopus laevis oocytes. The primary AbLV-specific protein synthesized is a polyprotein corresponding in molecular weight and immunological properties to a previously described p15 and p12 containing 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells. In contrast, translation of woolly monkey sarcoma virus genomic RNA resulted in symthesis of a 55,000-molecular-weight polyprotein consisting of woolly helper virus p30, p15, and p12. These findings demonstrate the value of the X. laevis oocyte in vitro system for studies of translational products of replication-defective transforming viruses and establish the virus-coded nature of the nonstructural component of the 110,000- to 130,000-molecular-weight polyprotein expressed in AbLV-transformed cells.     

Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants.

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.     

Functional role of arginine 302 within the lactose permease of Escherichia coli.

Within the lactose permease, an arginine residue is found on a transmembrane segment at position 302. Based upon the effects of mutations at or in the vicinity of Arg-302, this residue has been implicated to be involved with H+ and/or sugar recognition. To further elucidate the role of this residue, we have substituted Arg-302 with serine, histidine, and leucine via site-directed mutagenesis. All three of these substitutions result in an impaired ability to transport galactosides as evidenced by their poor growth on minimal plates supplemented with lactose or melibiose. Furthermore, in vitro transport assays revealed substantial alterations in the kinetic constants for downhill lactose transport. The wild-type strain exhibited a Km for lactose transport of 0.30 mM and a Vmax of 267 nmol of lactose/min.mg of protein. The Ser-302, His-302, and Leu-302 were observed to have Km values of 0.18, 2.3, and 2.8 mM, and Vmax values of 11.6, 56.4, and 22.0 nmol of lactose/min.mg of protein, respectively. In uphill transport assays, all three mutants were unable to accumulate beta-methyl-D-thiogalactoside. However, both the Ser-302 and His-302 mutants were able to accumulate lactose against a concentration gradient. During H+ transport assays, all three mutants were shown to transport H+ in conjunction with thiodigalactoside. In addition, the Ser-302 and His-302 strains exhibited small alkalinizations upon the addition of lactose. However, for the Leu-302 mutant, the addition of lactose did not result in a significant level of H+ transport. Finally, experiments were conducted which were aimed at measuring the ability of the mutant permeases to catalyze an H+ leak. In this regard, a comparison was made between the wild-type and mutant strains concerning their steady state pH gradient and their rates of H+ influx following oxygen pulses. The results of these experiments suggest that mutations at position 302 cause a sugar-dependent H+ leak.     

Normal expression of thymidine kinase and O6-methylguanine-DNA methyltransferase in cultured fibroblasts from individuals with hereditary galactokinase deficiency

Expression of the enzymes galactokinase, thymidine kinase, and O⁶-methylguanine-DNA methyltransferase is occasionally coordinately regulated in human cell lines. We have measured the activities of these three enzymes in extracts of fibroblasts from individuals with hereditary galactokinase deficiency. These cells do not express measurable galactokinase activity. The levels of O⁶-methylguanine-DNA methyltransferase were in the normal range in cells from three galactokinase-deficient individuals. The activity of thymidine kinase in the affected cells was in the normal range for two of the three individuals. The reduced thymidine kinase activity in the third individual reflected the extremely poor growth of the cells in culture. Immortalization of one galactokinase-deficient cell line resulted in loss of O⁶-methylguanine-DNA methyltransferase activity, but the galactokinase and thymidine kinase levels remained unchanged. The data indicate that the loss of galactokinase activity in these individuals is the consequence of an alteration of gene expression which does not involve coordinate silencing with the thymidine kinase and methyltransferase loci.     

Regulation of stem elongation in chinese cabbage by inflorescence removal and application of growth regulators

The effect of inflorescence removal on stem elongation in Chinese cabbage cv. Spring A was studied. Removal of the inflorescence before its visibility, or upon its appearance but before the beginning of bolting (stages 1–3), markedly reduced the stem length. Removal after the beginning of bolting (stage 5) had no effect on stem length.Application of GA₃ to the treated plants partially or fully restored the elongation of the flowering stem, whereas paclobutrazol inhibited the elongation of the treated, as well as the control stems. Indole-3-acetic acid (IAA) or kinetin was ineffective in restoring stem elongation of the plants from which the inflorescence had been removed. Inflorescences at stages 1–2 were found to secrete about 10 times more gibberellic acid (GA)-like activity compared with control apices or inflorescences at stage 5.It is suggested that the developing inflorescence is the major source of GAs which control stem elongation. However, shortly after the appearance of the inflorescence at the onset of bolting, stem elongation is no longer dependent on GAs derived from the apical inflorescence but require GAs from other sources.Contribution from the Agricultural Research Organization, The Volcani Center Bet Dagan, Israel No. 2218-E, 1987 series.     

Enhanced microvascular permeability of PMA-induced acute lung injury is not mediated by cyclooxygenase products.

Products of cyclooxygenase activity have been proposed to mediate the pulmonary hypertension and increased microvascular permeability associated with phorbol myristate acetate- (PMA) induced acute lung injury. Previously, we reported that thromboxane (Tx) does not mediate PMA-induced pulmonary hypertension in intact anesthetized dogs. In the present study, PMA was administered to isolated canine lungs perfused with autologous blood at constant flow to investigate a possible role for Tx in the PMA-induced increase in microvascular permeability. Changes in permeability were assessed by determining changes in the capillary filtration coefficient (Kfc). In lobes pretreated with papaverine to prevent PMA-induced increases in pulmonary vascular resistance, Kfc increased from a baseline value of 0.2 +/- 0.03 to 1.5 +/- 0.29 ml.min-1.cmH2O-1.100 g wet lobe wt-1 (P < 0.01) 30 min after PMA (5.8 x 10(-8) M, n = 10). Concomitantly, TxB2, the stable metabolite of TxA2, increased from 138 +/- 44 to 1,498 +/- 505 pg/ml (P < 0.05) in the blood. Both the selective Tx synthase inhibitor, OKY-046 (7 x 10(-4) M, n = 6), and the cyclooxygenase inhibitor, indomethacin (10(-4) M, n = 7), prevented the PMA-induced increase in TxB2, but neither compound attenuated the PMA-induced increase in Kfc. ONO-3708 (10(-6) M), a selective prostaglandin (PG) H2/TxA2 receptor antagonist, prevented the vasoconstriction resulting from administration of U-46619, a stable PGH2/TxA2 receptor agonist, but it did not prevent the PMA-induced increases in Kfc (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)