Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 μg/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na⁺, K⁺, Mg²⁺ or Ca²⁺ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

Effect of age on contractile response to angiotensin II in rat aorta

The effects of aging on contractile response to angiotensin II and tachyphylaxis to it were investigated using aortic strips from rats aged 1.5, 4 and 22 months. Whether the endothelium was present or not, the contractile response to angiotensin II was greater and tachyphylaxis to it was less in 1.5-month-old rats than in 4- and 22-month-old rats. The differences between 4- and 22-month-old rats were not significant. Removal of the endothelium enhanced angiotensin II-induced maximal contraction and depressed the tachyphylaxis, these endothelial effects being greater in 4- rather than in 1.5-month-old rats. When the contractile force of angiotensin II was adjusted to a similar level for 1.5- and 4-month-old rats, the endothelial effect on the tachyphylaxis was greater in the 4-month-old rats, but no significant difference was noted in the endothelial effect on the contractile force. These results suggest that during growth, the contractile response of rat aorta to angiotensin II decreases while the endothelial effect on it increases.     

Crystallization of a new class of microbial ribonuclease from Rhizopus niveus

Crystals of ribonuclease Rh, a new class of microbial ribonuclease from Rhizopus niveus, were obtained from polyethylene glycol 8000 solution by a vapour diffusion technique in the hanging drop mode. Two crystal forms, type I and type II, were obtained from the same droplet solution. Both forms belong to the space group P2(1)2(1)2(1), but their cell dimensions are markedly different: a = 68.3 A, b = 73.0 A, c = 50.0 A for type I and a = 67.5 A, b = 72.3 A, c = 44.2 A for type II. The type I crystals diffract beyond 2.0 A resolution and are suitable for X-ray structure analysis at high resolution.     

Life cycle of Hysterothylacium haze (Nematoda: Anisakidae: Raphidascaridinae)

Natural infections with Hysterothylacium haze in the Japanese common goby, Acanthogobius flavimanus, were observed in detail. In gobies in which no worm eggs were deposited, second-stage larvae were found in the digestive tract wall, and third-stage larvae occurred in the digestive tract wall, mesentery, and body cavity, whereas fourth-stage larvae and adults were found in the body cavity. This stage-habitat relationship demonstrates the infectivity of second-stage larvae to the goby and the larval migration. In heavily infected gobies, eggs and all worm stages, from hatched second-stage larvae to adults, often were found together in the body cavity of one individual host, suggesting that hatched second-stage larvae can develop in the body cavity. It was shown experimentally that H. haze develops to the second stage in the egg and does not hatch spontaneously. When a goby was fed the viscera of heavily infected gobies containing eggs and various stages of worms or artificially incubated eggs containing second-stage larvae, second- and third-stage larvae were recovered from the digestive tract wall, and fourth-stage larvae and adults were found in the body cavity. When polychaetes or crustaceans were placed in contact with infected goby viscera or incubated eggs, only second-stage larvae were recovered from the body cavity of the invertebrates. The experimental results were consistent with observations on natural infections and indicate that the direct life cycle of H. haze may involve invertebrates as transport hosts.     

Fine structure of ependymal cells in the median eminence of the frog and mouse revealed by freeze-etching

Summary Freeze-etched preparations of the ventricular surfaces of ependymal cells clearly reveal the presence of pinocytotic vesicles opening into the third ventricle and large vacuoles formed by broad cell projections. The density of the vesicular openings is approximately 20 per m². The ependymal cells in the median eminence of the frog are adjoined by tight junctions comprised of five to eight interconnected junctional strands, whereas near the median eminence in the mouse only one to two such strands form the tight junction of the ependymal cells. Gap junctions between the adjacent ependymal cells are detected near the median eminence in the mouse but not in the frog.This study was supported in part by a grant from the Japanese Ministry of Education (No. 067670)     

Synthesis of some glycodipeptides containing hydroxyamino acids, and their stabilities to acids and bases

The following new compounds were prepared and characterized: N-benzyl-oxycarbonyl-O-(tetra-O-acetyl-β-D-glucopyranosyl)-N-glycyl-L-serine methyl ester (1) and L-threonine methyl ester (2), N-benzyloxycarbonyl-O-(β-D-glucopyranosyl)-N-glycyl-L-serine amide (3), N-benzyloxycarbonyl-O-(β-D-glucopyranosyl)-N-glycyl-L-threonine methyl ester (4) and L-threonine amide (5), N-benzyloxycarbonyl-O-(tri-O-acetyl-2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-N-glycyl-L-serine methyl ester (6), and N-benzyloxycarbonyl-O-(2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-N-glycyl-L-serine amide (7). Although various modifications of the Koenigs-Knorr synthesis were used, the best, over-all yields of the deacetylated dipeptide derivatives were only 5–10%. Although the products are alkali-labile, deacetylation was accomplished with methanolic ammonia. Of the deacetylated products, the threonine derivatives (4 and 5) were more rapidly hydrolyzed by acids than phenyl β-D-glucopyranoside, which in turn was more rapidly cleaved than the serine derivatives (3 and 7). The stabilities of 3, 4, 5, and 7 to sodium hydroxide and sodium borohydride were similar, and essentially complete β-elimination of the glycosyl residue occurred for the amide derivatives (3, 5, and 7). For the ester derivative 4, pH 9 was optimal; above this pH, ester hydrolysis was more rapid than β-elimination, and the resulting carboxyl derivatives did not undergo β-elimination. Under optimal conditions with sodium borohydride, the β-elimination reaction was complete, but the corresponding alanine and α-aminobutyric acid residues were not formed; presumably reductions to the amino alcohols occurred. A mechanism for the β-elimination is proposed.     

Stereospecific reduction of the butenolide in strigolactones in plants

Reductive metabolism of strigolactones (SLs) in several plants was investigated. Analysis of aquaculture filtrates of cowpea and sorghum each fed with four stereoisomers of GR24, the most widely used synthetic SL, revealed stereospecific reduction of the double bond at C-3′ and C-4′ in the butenolide D-ring with preference for an unnatural 2′S configuration. The cowpea metabolite converted from 2′-epi-GR24 and the sorghum metabolite converted from ent-GR24 had the methyl group at C-4′ in the trans configuration with the substituent at C-2′, different from the cis configuration of the synthetic H₂-GR24 reduced with Pd/C catalyst. The plants also reduced the double bond in the D-ring of 5-deoxystrigol isomers with a similar preference. The metabolites and synthetic H₂-GR24 stereoisomers were much less active than were the GR24 stereoisomers in inducing seed germination of the root parasitic weeds Striga hermonthica, Orobanche crenata, and O. minor. These results provide additional evidence of the importance of the D-ring for bioactivity of SLs.     

The amino acid sequence of the β-subunit: Of porcine enterotoxigenic Escherichia coli enterotoxin — Analysis and comparison with literature data

Abstract The amino acid composition and sequence of the β-subunit of heat-labile enterotoxin (LT) purified from a porcine (LT_p) strain, WT-1, of enterotoxigenic Escherichia coli was analysed, and the result was compared with that reported by Dallas and Falkow [Nature 288 (1980) 499-501] who deduced the amino acid sequence of LT_p from data on the DNA sequence of a porcine strain, EWD299. The purified β-subunit of the LT_p of WT-1 was carboxymethylated, succinylated, digested with chymotrypsin and subjected to high performance liquid chromatography (HPLC). The amino acid composition of the peptide peaks from the column were analysed and compared with the data reported by Dallas and Falkow. Only one fraction differed in amino acid composition from that reported, containing lysine instead of methionine. This fraction was found to consist of two peptides with the sequences Lys-Ser-Gly-Glu-Thr-Phe and Arg-Ile-Thr-Tyr. The former peptide is reported to have the sequence Met-Ser-Gly-Glu-Thr-Phe. Thus, the amino acid at position 43 from the N terminus of the β-subunit of LT_p is lysine, not methionine as reported. This is the first report which studied the amino acid sequence of LT_p analysed by protein toxin itself, not by DNA sequence analysis.     

Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61--77 from Escherichia coli tRNAfMet.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.     

Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. I: Topology of bovine adrenal cortex mitochondria in the orthodox configuration

Bovine adrenal cortex mitochondria examined by electron microscopyin situ orin vitro in 0·25 M sucrose have an unusual cristal membrane structure. The cristae usually appear as unconnected vesicles within a double membrane system. A few of the vesicles appear to be attached to the inner boundary membrane or to one or more other vesicles. The configuration of such mitochondria will be defined as the orthodox configuration. In this communication we will provide evidence that the inner membrane is not composed of multiple vesicles, but is one continuous membrane with tubular invaginations, and that these invaginations alternately are ballooned out and squeezed down. A mechanism has been proposed to account for the differentiated structure of the cristae of adrenal cortex mitochondria.