Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from Staphylococcus aureus and its relationship to AAD(3") (9)

Summary The nucleotide sequence of the spc determinant of the Staphylococcus aureus transposon Tn554 has been determined. This gene encodes a spectinomycin adenyltransferase, AAD(9), that mediates resistance to spectinomycin but not to streptomycin. The sequence predicts a 260 amino acid protein of molecular weight 28,943. A spectinomycin-sensitive mutant (spc-1) contains a GA transition resulting in substitution of threonine (ACA) for alanine (GCA) at residue 165. The predicted amino acid sequence is 36% homologous to that of a widely distributed, gramnegative streptomycin/spectinomycin adenyltransferase, AAD(3) (9), specified by the aadA determinant (Holingshead and Vapnek 1985).     

Taxonomic studies of the genus Hypoxis in East Africa

East African material of the genus Hypoxis L. has preliminarily been divided into the heterogenous, probably apomictic H. obtusa Burch- complex (2n = 40–50, ca. 75, 76, ca. 85, >86, ca. 92, ca. 98, ca. 108, 130–135, 160–200) and 5 rather homogenous species: H. angustifolia Lam. (2n = 14, 28), H. goetzei Harms (2n = ca. 62), H. kilimanjarica Bak., H. malosana Bak. (2n = 14) and H. macrocarpa Holt & Staubo sp. nov. H. kilimanjarica is divided into ssp. kilimanjarica and ssp. prostrata Holt & Staubo ssp. nov.     

Characteristics of bacterium GB-2, a presumptiveCytophaga species with novel immunoregulatory properties

A Gram-negative, psychrophilic bacterium, designated GB-2, was isolated from fetal calf serum and analyzed for its morphological, physiological, and biochemical characteristics. Gliding motility, sensitivity to actinomycin D, the presence of the pigment flexirubin and cytochrome c, growth on a variety of carbohydrates, the production of acid fermentation products, and a 34.9 mol% guanine+cytosine (G+C) content of bacterial DNA indicate GB-2 to be a member either of the generaCytophaga orFlexibacter. Growth of GB-2 was optimized in a simple defined medium to facilitate isolation and characterization of bacterial products. Liquid growth of GB-2 resulted in the release of significant quantities of a macromolecule free of both endotoxin and protein into the growth supernatant, which activated the proliferation of murine lymphocytes. The relationship between this bacterium and its end-products to other species of theCytophaga/Flexibacter group is discussed.     

Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences

S-Adenosyl-L-homocysteine was used to inhibit the methylation of carboxylic acid residues of membrane proteins in intact human erythrocytes. Incubation of erythrocytes for 24 h with 5 mM each of adenosine and L-homocysteine resulted in the intracellular accumulation of S-adenosyl-L-homocysteine and substantially inhibited membrane protein carboxyl methylation. From the degree of inhibition and from the observed turnover of methylated proteins, we estimate that the number of protein methyl esters in cells incubated with adenosine and L-homocysteine for 20 h is less than 20% that of cells incubated without these inhibitors. No significant differences in the physical deformability properties of the membrane of these hypomethylated cells were detected. However, there was a small but significant (p less than 0.001) increase in the amount of membrane protein D-aspartyl residues in these cells compared to control cells. These observations are consistent with the hypothesis that methylation of membrane proteins at D-aspartyl residues may result in the selective removal or repair of these uncommon residues.     

A limited region within hen egg-white lysozyme serves as the focus for a diversity of T cell clones

The C57BL/6 (H-2b) mouse is a nonresponder to hen egg-white lysozyme (HEL) injected i.p., owing to a T suppressor cell-inducing determinant at the amino-terminal region. After immunization with a 93-amino acid fragment (a.a. 13-105) of HEL lacking this determinant, all clones from two independently derived C57BL/6 T cell lines were found to be specific for epitopes within a subregion of peptide 74-96. Three specificity patterns for the clones could be defined on the basis of cross-reactivities with only two other species variant lysozymes. Reactivities of all three specificity groups was consistent with the serine to threonine substitution at position 91, although reactivity of one of the groups could be affected by substitutions at position 84. The results confirm at the clonal level that even for distantly related antigens, only limited regions are recognized by T cells. They are consistent with the notion that specific sites on the antigen capable of interaction with Ia molecules lead to dominance of certain regions for T cell reactivity. Moreover, the diversity in specificity among clones suggests that the limiting feature of T cell responsiveness is not a lack of available T cells in the repertoire directed against a single antigenic site.     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

Vaccination against schistosomiasis in mice with killed schistosomula without adjuvant

Attempts to develop a killed vaccine against schistosomiasis have generally resulted in failure. There are two recent reports, but unfortunately, harsh adjuvants were used in conjunction with the antigenic materials. In our laboratory, a killed vaccine was developed by freezing (-196 degrees C) and thawing the schistosomula of S. mansoni. The use of such a preparation without adjuvant was effective in vaccinating mice. A worm reduction of 36.4-41.1% was achieved by one vaccinating injection, a 60.2% worm reduction by 3 injections, and a 63.7-66.0% reduction by 5 injections. The sequence of the development and the expression of the immune reactions were similar to those previously found in hosts immunized with highly X-irradiated schistosome organisms. Delayed hypersensitivity was demonstrated in histological sections of the skin in the challenged mice after one vaccination, showing that an adjuvant was not necessary to initiate the induction of cellular immunity.     

The interaction of cadmium with calcium and cisplatin with chloride

Changes in the locomotor rate of the ciliateTetrahymena pyriformis were used to quantitatively evaluate chemical interactions produced by: cadmium in combination with varying amounts of calcium, andcis-dichlorodiammineplatinum (II) (cisplatin) with varying amounts of sodium chloride. Cadmium (as CdCl₂) produces a measurable decline in the locomotor rate of the cells. Cadmium's detrimental effect can be reduced by the addition of calcium (as CaCl₂) in combination with cadmium. At a ratio of 30∶1 (calcium: cadmium), cadmium's negative effect upon motility is essentially nullified. It is suggested that the “protective” action afforded by calcium stems from the chemical similarity of the two cations and their involvement/competition for molecular sites responsible for the energy release and/or delivery of ciliary activity. Cisplatin will also effect a reduction in ciliary activity. However, the interaction between cisplatin, sodium chloride, and the cell appears more complex than that found with cadmium-calcium. At the lower range of chloride (as NaCl) used in this study, increased chloride concentration produces an increase in cisplatin's action against ciliary activity. At the higher levels, the chloride reduced cisplatin's negative effects. It is suggested that the increases in cisplatin's effects are caused by mass chemical action of increased chloride, which increases the concentration of the nonpolar cisplatin. The reduced effects found with the higher concentrations of sodium chloride may be because of the presence and action of elevated NaCl in/on the cell. This study clearly demonstrates differences in biologically relevant chemical interactions occurring with the two sets: cadmium-calcium and cisplatin-chloride.     

Carboxyl methylation of human erythrocyte band 3 in intact cells. Relation to anion transport activity.

The anion transport protein of the human erythrocyte membrane, band 3, is reversibly methylated by an endogenous protein carboxyl methyltransferase. The physiological consequence of this modification was studied by measuring the rate of phosphate transport by intact erythrocytes incubated under conditions where protein methylation reactions are inhibited. No change in phosphate transport was detected when cells were treated with either methionine-free media or cycloleucine, whereas cells incubated with adenosine and homocysteine thiolactone displayed a marginally slower rate of transport, which was not reversed by subsequent remethylation of the membrane proteins. These results suggest that erythrocyte protein carboxyl methylation does not directly regulate this activity of band 3.