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 effect of diabetes mellitus on aortic prostanoid synthesis and serum cholesterol levels in the rat fed a high cholesterol diet

The effect of diabetes mellitus on serum cholesterol and aortic microsomal prostanoid synthesis was studied in cholesterol fed male Lewis rats. Normal, diabetic and diabetic rats treated with pancreatic islets were divided into three diet subgroups, control diet, control +2% cholesterol for 8 weeks and control +2% cholesterol diet for 16 weeks. Serum glucose levels were elevated three-fold in the diabetic group compared to normal. Treatment with islets restored serum glucose to normal levels in diabetic rats. The 2% cholesterol diet did not significantly alter serum glucose levels in any of the groups. Body weights in the diabetic group were significantly lower than normal or diabetic rats treated with islets. Feeding 2% cholesterol for 16 weeks significantly increased weight in normal and islet treated diabetic rats but not in the diabetic group. Aortic microsomal prostanoid synthesis was similar in all experimental groups with 6-keto-PGF1 alpha (PGI2 metabolite) being the major product synthesized in all groups. Aortic microsomal prostanoid levels were not altered by the 2% cholesterol diet. Serum cholesterol levels increased 14-fold in the diabetic group which returned to the normal level in the diabetic animals treated with islets. These data show that diabetes does not alter aortic microsomal prostanoid levels in the rat. However, diabetes significantly increased serum cholesterol levels which were reversed by islet transplantation.     

Increased shikonin production inLithospermum erythrorhizon suspension cultures within situ extraction and fungal cell treatment (elicitor)

Summary The effect ofin situ extraction and elicitor treatment on shikonin production was studied with the suspension cultures ofLithospermum erythrorhizon. Shikonin concentration of 60 mg/L was achieved by the use of both techniques which was 24 times higher than that of control culture, and 65 times higher in terms of shikonin productivity. The host-pathogen effect of elicitor treatment andin situ extraction for product removal were effective for shikonin production.     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Production of a polysaccharide, methylan, in Methylobacterium organophilum by controlling ammonium ion

Summary Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.     

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.     

New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

The carboxylate type siderophore rhizoferrin and its analogs produced by directed fermentation

Summary Rhizoferrin is a novel carboxylate-type siderophore which has recently been isolated fromRhizopus microsporus and other fungi of the Mucorales (Zygomycetes). The present investigation shows that a variety of rhizoferrin analogs can be produced by directed fermentation. Thus both the diaminobutane backbone and the citric acid side chains of rhizoferrin have been substituted by diamine and citric acid analogs added to the culture medium. The new ligands as well as their iron complexes have been characterized by physicochemical methods. Conditions of precursor incorporation and implications for the biosynthesis of the new siderophores are discussed.