Use of tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in porstaglandin and thromboxane radioimmunoassays. Assays for PGF_2α, 15-keto-13, 14-dihydro-PGF_2α, TXB₂ and 15-keto-13, 14-dihydro-TXB₂ were evaluated in comparative studies using either these heterologous ligands or the corresponding homologus tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer.Three biological studies were also conducted, viz. study of the release of TXB₂ during collagen induced platelet aggregation, of 15-keto-13, 14-dihydro-TXB₂ during guinea pig pulmonary anaphylaxis, and of PGF_2α (measured as 15-keto-13, 14-dihydro-PGF_2α in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay.Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.     

Tubular structures associated with the endothelial endoplasmic reticulum in glomerular capillaries of Rhesus monkey and nephritic man

Summary Round bodies, tubular profiles and crystalline images have been studied by electron microscopy in the endothelium of seven normal young Rhesus monkeys and in the renal glomerular endothelium of two nephritic human patients. The crystalline images are most frequently formed by aggregation of round bodies, 200–240 Å in diameter. In Rhesus monkeys a variety of crystalline images are seen. On the contrary, in nephritic patients round bodies and tubular profiles are rare and less organized. In the glomerular endothelium of two normal men they were not found.The results obtained suggest that the round bodies, the tubular profiles and the crystalline images result from sectioning of a system of undulating tubules associated with the smooth endoplasmic reticulum. In nephritic patients the formation of such a tubular system could represent a change taking place within the affected cells as a pathologic response to the disease.This work was supported by U.S. Public Health Service (N.H.I.), Grant AI-04527-03-04, and by the Consiglio Nazionale delle Ricerche (C.N.R.), Contributo 115/0815/0-1365. The Authors are greatly indebted to Miss Hermina Spiele for skilful technical assistance.     

Antimicrobial activity of clove oil dispersed in a concentrated sugar solution

Essential oil of clove, dispersed (0.4%v/v) in a concentrated sugar solution, had a marked germicidal effect against various bacteria and Candida albicans. Staphyloccus aureus (five strains), Klebsiella pneumoniae, Pseudomonas aeruginosa, Clostridium perfringens , and Escherichia coli inoculated at a level of 10⁷ cfu/ml, and C. albicans (inoculum 4.0×10⁵ cfu/ml) were killed (< 99.999%) after 2–7 min in a laboratory broth supplemented with 63% (v/w) of sugar, and containing 0.4% (v/w) of essential oil of clove. Added organic matter (i.e. human or bovine serum) did not impair its antimicrobial activity.
Sugar was not necessary for the antimicrobial activity of clove oil, but the concentrated sugar solution provided a good vehicle for obtaining an oil dispersion that is relatively stable for certain practical applications.     

Lead as an inductor of some morphological and functional changes in synaptosomes from rat brain

Summary 1. The effect of lead (in vivo) on the uptake of GABA, dopamine, and histidine as a precursor of histamine in synaptosomes obtained from chronically lead-treated rats was studied.2. Lead decreased the uptake of GABA, increased the uptake of dopamine, and did not change the uptake of histidine. These effects were independent of calcium concentration.3. Lead administration to the rat changed the morphology of the synaptosomes, as manifested in the decreased number of synaptic vesicles and disturbed mitochondrial structure.4. The results suggest the existence of several mechanisms of lead toxicity on uptake, related to individual neurotransmitters, which are not necessarily connected with a Pb²⁺/Ca²⁺ interaction.     

Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid

The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa pro-FatB precursor protein.     

Further structural studies of zosterine

Traces of either ferrous or ferric salts greatly increase the rate of the stepwise degradation of reducing sugars by alkaline hydrogen peroxide, as measured by formation of formic acid; addition of larger proportions of iron salts causes relatively smaller effects. The results showed that, unless unusually strict precautions are taken to exclude traces of iron, the free-radical cleavage of the hydroperoxide adducts of reducing sugars is far more rapid than the ionic cleavage. The catalytic effect of iron salts is counteracted by addition of magnesium salts. With d-glucose, inhibition of the catalytic effect of iron by magnesium depends on both the magnesium-iron ratio and the concentration at a given ratio. Measurements with various molar proportions of the salts indicated that a magnesium-iron complex, containing six atoms of magnesium to one of iron, is formed. Presumably, removal of iron by formation of this complex inhibits the free-radical degradation of hydroperoxide adducts. In marked contrast to the results obtained with reducing sugars, the degradation of potassium glyoxylate and of glyoxal by alkaline hydrogen peroxide is extremely rapid, and not catalyzed by iron or inhibited by magnesium. The results are in accord with an ionic, rather than a free-radical, cleavage of the hydroperoxide adducts of these compounds. The rapidity of the ionic reaction may be attributed to the ready availability of an electron pair from the adjoining carbon atom.     

Isolation and Characterization of a Novel Bacteriophage φ4D Lytic Against Enterococcus faecalis Strains

In recent years, Enterococcus faecalis has emerged as an important opportunistic nosocomial pathogen capable of causing dangerous infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages have very effective bactericidal activity and several advantages over other antimicrobial agents and so far, no serious or irreversible side effects of phage therapy have been described. The objective of this study was to characterize a novel virulent bacteriophage φ4D isolated from sewage. Electron microscopy revealed its resemblance to Myoviridae, with an isometric head (74 ± 4 nm) and a long contractile tail (164 ± 4 nm). The φ4D phage genome was tested using pulsed-field gel electrophoresis and estimated to be 145 ± 2 kb. It exhibited short latent period (25 min) and a relatively small burst size (36 PFU/cell). Tests were conducted on the host range, multiplicities of infection (MOI), thermal stability, digestion of DNA by restriction enzymes, and proteomic analyses of this phage. The isolated phage was capable of infecting a wide spectrum of enterococcal strains. The results of these investigations indicate that φ4D is similar to other Myoviridae bacteriophages (for example φEF24C), which have been successfully used in phagotherapy.     

Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

Saccharomyces cerevisiae Msl5 orchestrates spliceosome assembly by binding the intron branchpoint sequence 5′-UACUAAC and, with its heterodimer partner protein Mud2, establishing cross intron-bridging interactions with the U1 snRNP at the 5′ splice site. Here we define the central Msl5 KH-QUA2 domain as sufficient for branchpoint RNA recognition. The 1.8 Å crystal structure of Msl5-(KH-QUA2) bound to the branchpoint highlights an extensive network of direct and water-mediated protein–RNA and intra-RNA atomic contacts at the interface that illuminate how Msl5 recognizes each nucleobase of the UACUAAC element. The Msl5 structure rationalizes a large body of mutational data and inspires new functional studies herein, which reveal how perturbations of the Msl5·RNA interface impede the splicing of specific yeast pre-mRNAs. We also identify interfacial mutations in Msl5 that bypass the essentiality of Sub2, a DExD-box ATPase implicated in displacing Msl5 from the branchpoint in exchange for the U2 snRNP. These studies establish an atomic resolution framework for understanding splice site selection and early spliceosome dynamics.