Structural gene isolation and prepeptide sequence of gallidermin, a new lanthionine containing antibiotic

Peptide antibiotics containing lanthionine and 3-methyllanthionine bridges, named lantibiotics are of increasing interest. A new lantibiotic, gallidermin, has been isolated from Staphyloccus gallinarum. Here we report the isolation of its structural gene which we name gdmA. In all lantibiotics so far studied genetically, three peptides can be formally distinguished: (i) the primary translation product, which we call the prepeptide; (ii) the propeptide lacking the leader sequence and (iii) the mature lantibiotic. Unlike the plasmid-coded epidermin, gdmA is located on the chromosome. The gdmA locus codes for a 52 amino acid residue prepeptide, consisting of an alpha-helical leader sequence of hydrophilic character, which is separated from the C-terminus (propeptide) by a characteristic proteolytic processing site (Pro-2 Arg-1 Ile1). Although pro-gallidermin differs from pro-epidermin (a recently isolated lantibiotic) only by a single amino acid residue exchange. Leu instead of Ile, the N-terminus of the prepeptide differs by an additional two exchanges.     

The perennial taxa ofCrucianella (Rubiaceae) in SW. Asia and their eco-geographical differentiation

The perennial taxa ofCrucianella in Asia form a coherent group, apparently diploid (x = 11) and outbreeding throughout, and should be placed into sect.Roseae. This Irano-Turanian group has its centre of diversity in the mountain systems south of the Caspian Sea and reaches with outposts NE. and E. Anatolia, NE. Iraq, S. Iran and C. Asia. Four species and 13 subspecies (within the polymorphicC. gilanica) are recognized, described (partly as new), and illustrated (Figs. 1–6). Conspectus, keys and distribution maps (Figs. 7 and 8) as well as plesio- and apomorphic character states and data on size of areas are provided (Table 1). There is an obvious correlation between more plesiomorphic taxa with smaller areas in the distribution centre of the group, and more apomorphic taxa with larger areas towards its periphery (Fig. 9). These findings are linked to a working hypothesis on the evolution of the group.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Physical Basis for Altered Stem Elongation Rates in Internode Length Mutants of Pisum

Biophysical parameters related to gibberellin (GA)-dependent stem elongation were examined in dark-grown stem-length genotypes of Pisum sativum L. The rate of internode expansion in these genotypes is altered due to recessive mutations which affect either the endogenous levels of, or response to, GA. The GA deficient dwarf L181 (ls), two GA insensitive semierectoides dwarfs NGB5865 and NGB5862 (Ika and Ikb, respectively) and the `slender' line L197 (la cry^[ill]), which is tall regardless of GA content, were compared to the wild-type tall cultivar, Torsdag. Osmotic pressure, estimated by vapor pressure osmometry, and turgor pressure, measured directly with a pressure probe, did not correlate with the differences in growth rate among the genotypes. Mechanical wall properties of frozen-thawed tissue were measured using a constant force assay. GA deficiency resulted in increased wall stiffness judged both on the basis of plastic compliance and plastic extensibility normalized for equal stem circumference. Plastic compliance was not reduced in the GA insensitive dwarfs, though Ika reduced circumference-normalized plasticity. In contrast, in vivo wall relaxation, determined by the pressure-block technique, differed among genotypes in a manner which did correlate with extension rates. The wall yield threshold was 1 bar or less in the tall lines, but ranged from 3 to 6 bars in the dwarf genotypes. The results with the ls mutant indicate that GA enhances stem elongation by both decreasing the wall yield threshold and increasing the wall yield coefficient. In the GA-insensitive mutants, Ika and Ikb, the wall yield threshold is substantially elevated. Plants possessing Ika may also possess a reduced wall yield coefficient.     

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.     

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.     

Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin.

The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.