The lack of an effect of furosemide on uterine prostaglandin metabolism in vivo

We determined the effect of 2 mg/kg intravenous furosemide on the production and metabolism of prostaglandin E2 in the utero-placental unit of pregnant dogs. Uterine venous prostaglandins E2 and 15-keto-13,14-dihydro E2 were measured by gas chromatography-mass spectrometry. Even though the dose of furosemide was adequate to effect a good diuresis, neither the production nor the metabolism of prostaglandin E2 by the uterus was altered by that dose of the drug. Using radioactive microspheres to measure hemodynamic parameters, we observed no change in uterine vascular resistance while renal vascular resistance decreased. Although the renal concentration of furosemide may be higher than the uteroplacental concentration, there is so far no evidence in vivo that usual doses of furosemide enhance the production or inhibit the metabolism of prostaglandin E2.     

Enhanced bactericidal activity of platelet β-lysin forE. coli in the presence of membrane perturbation

Inhibition by sodium chloride of the growth of 19 strains ofLegionella pneumophila and of 10 strains of otherLegionella spp. was studied. Results from growth in buffered -ketoglutarate cysteine yeast extract (BAYE) broth containing 0 to 2.0% sodium chloride indicated that 15/19 laboratory strains ofL. pneumophila were capable of growing in 1.0% to 1.5% sodium chloride, whereas 4 strains ofL. pneumophila and 10 strains of 6 other species were not.L. micdadei andL. longebeachae were the most inhibited in BAYE broth, growing only in concentrations of 0.5% sodium chloride. These in vitro studies indicate thatL. micdadei andL. longbeachae might be differentiated from other species by their low tolerance to salt in BAYE broth, and thatL. pneumophila may be more tolerant to salt concentrations found in brackish water environments.     

Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery.

We used perfused rat livers to investigate the role of endosomes versus lysosomes in the hydrolysis of endocytosed material. When perfusions were performed at 37 degrees C with 125I-asialoorosomucoid, 125I-galactosylated albumin, or 125I-mannosylated albumin, there was a 15-min lag before trichloroacetic acid-soluble degradation products were detected. Furthermore, no hydrolysis was detected at 16 degrees C, indicating that there was no significant prelysosomal degradation of these proteins. Since detection by this method depends on extensive hydrolysis, we subsequently used three small synthetic molecules from which fluorescent products are generated by a single cleavage. These were 4-methylumbelliferyl sulfate, 4-methylumbelliferyl phosphate, and 4-methylumbelliferyl-beta-D-glucosaminide, which are substrates for aryl sulfatase, acid phosphatase, and beta-hexosaminidase, respectively. Using the first two compounds, hydrolysis was detected after 3 min at 37 degrees C and still occurred, albeit to a reduced extent, at 16 and 4 degrees C. This indicates that aryl sulfatase and acid phosphatase are active prelysosomally. We found a different result with 4-methylumbelliferyl-beta-D-glucosaminide. At 37 degrees C, there was a greater than 15-min lag before hydrolysis products were measured; furthermore, hydrolysis ceased at 16 degrees C, indicating that beta-hexosaminidase is active lysosomally. Taken together, these findings show that there is selective activation and/or delivery of hydrolases along the endocytic pathway.     

Sequential processing of epidermal growth factor in early and late endosomes of rat liver.

We have used isolated perfused rat livers to examine the intracellular processing of 125I-epidermal growth factor (EGF) and to determine where in the endocytic pathway the hydrolases which degrade EGF are acting. Following uptake of 125I-EGF at 37 or 16 degrees C, subcellular fractions enriched in endosomes and lysosomes were isolated, and their 125I-EGF content was examined by reverse-phase high performance liquid chromatography. Three forms of EGF processed at their carboxyl termini are generated in endosomes. At 37 degrees C, EGF is first processed in early endosomes by a carboxypeptidase B-like protease and is further processed in late endosomes by a trypsin-like protease and then a carboxypeptidase B-like protease. At 16 degrees C, entry of EGF into late endosomes is slowed, and only the first processed form is generated over 60 min. Longer perfusions (180 min) at 16 degrees C result in some processing (7%) by proteases found in late endosomes. EGF-horseradish peroxidase cytochemistry confirmed that the additional processing detected at 180 min correlated with movement of EGF from tubulovesicular to multivesicular endosomes. These results, combined with in vitro incubations of EGF in isolated endosomal and lysosomal fractions, suggest that different proteases are active at selective points in the endocytic pathway and that the full complement of proteases needed for complete degradation of EGF is active only in lysosomes.     

Computer modeling 16 S ribosomal RNA

A three-dimensional structure for 16 S RNA has been produced with a computer protocol that is not dependent on human intervention. This protocol improves upon traditional modeling techniques by using distance geometry to fold the molecule in an objective and reproducible fashion. The method is based on the secondary structure of RNA and treats the molecule as a set of double-stranded helices that are linked by flexible single-strands of variable length. Data derived from chemical cross-linking studies of 16 S RNA and tertiary phylogenetic relationships provide the constraints used to fold the molecule into a compact three-dimensional form. Possibly subjective evaluation of the input data are transformed into verifiable quantitative parameters. Relationships based on general locations within the 30 S subunit or on protein-RNA interactions have been specifically excluded. The resolution of the model exceeds that of electron micrographs and approaches that obtained in preliminary X-ray crystal structures. The model size of 245 x 190 x 140 A is compatible with that of the 30 S subunit as determined by electron microscopy. The volume of the model is 1.87 x 10(6) A which is similar to that of the small subunit in a preliminary X-ray crystal structure. The radius of gyration of the model structure of 76 A is intermediate to that seen for partially denatured and fully folded 16 S RNA. Computer graphics are used to display the results in a manner that maximizes the opportunities for human visual interpretation of the models. A format for displaying the structures has been developed that will make it possible for researchers who have not devoted themselves to ribosomal modeling to comprehend and make use of the information that the models embody. On this basis the computer-generated models are compared with models developed by other researchers and with structural data not included in the folding parameter data set.     

The influence of ionic composition on the distribution of nematode species in springs of the province of Granada (Spain)

The ionic composition of 38 mineral springs in the province of Granada (Spain), and the distribution of 45 species of nematodes belonging to orders Monhysterida, Araeolaimida, Chromadorida and Enoplida were examined. Water chemistry is used to make two diagrams representing anionic and cationic composition. Diagrams for anionic composition (given the greater variance seen in the springs considered) are used to illustrate the distribution of individual species. The results obtained from species distribution and the correlation between species made it possible to group species which could be associated with springs where each of the anions considered predominated. A greater number of species groups was found to inhabit springs in which chloride concentrations was less than 50% of the total concentration of anions.     

Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.     

Evidence for pulmonary release of 5-hydroxyeicosatetraenoic acid (5-HETE) during endotoxemia in unanesthetized sheep

Leukocyte trapping in the pulmonary circulation may be an important component of the lung vascular injury response to endotoxin, but mediators of the pulmonary leukostasis and increased lung vascular permeability are unknown. The leukocyte 5-lipoxygenation pathway of arachidonic acid metabolism yields highly biologically active products including leukotrienes C₄ and D₄ (formerly slow reacting substance of anaphylaxis) and the potent chemotaxin, leukotriene B₄. A major product of 5-lipoxygenation is 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), for which a sensitive, stable isotope dilution assay employing combined gas chromatography-mass spectrometry is available. This assay was used to test the hypothesis that 5-lipoxygenation products might participate in pulmonary vascular responses to endotoxin. We measured 5-HETE concentrations in lung lymph at three intervals during endotoxemia in unanesthetized sheep. Concentrations of 5-HETE in lung lymph exceeded those in aortic blood plasma. Lymph 5-HETE concentrations increased from 1.7±0.3 (mean ± SEM, N = 7) ng/ml during baseline to peak values of 6.1±1.8 ng/ml (p < 0.05) during the hours after endotoxemia and preceeding the steady state increased lung vascular permeability response. During the increased permeability steady state from 240 to 270 minutes after endotoxin, lymph 5-HETE concentrations (1.4±0.3 ng/ml) and lymph 5-HETE flow (i.e., 5-HETE concentration x lung lynph flow rate) returned to baseline values. Although these observations are consistent with the hypothesis that 5-lipoxygenation products participate in the pulmonary vascular injury response to endotoxin, lymph 5-HETE concentrations did not correlate with any of the other experimental measurements. It may be only coincidence that the increase in lymph 5-HETE concentrations appeared contemporaneous with the onset of lung vascular injury.     

The site of VX2 tumor transplantation affects the development of hypercalcemia in rabbits

The relationship between plasma levels of 15-keto-13, 14-dihydro-prostaglandin E₂ (15K-H₂-PGE₂) and serum calcium levels was studied in nontumor-bearing rabbits and in rabbits bearing the VX₂ carcinoma intramuscularly and intra-abdominally. The plasma levels of 15K-H₂-PGE₂ in the two groups of tumor-bearing animals did not vary significantly but was several fold greater than in nontumor-bearing rabbits. Rabbits bearing the VX₂ carcinoma intramuscularly developed hypercalcemia between the second and third week after implantation of neoplastic tissue and remained hypercalcemic until they expired. The serum calcium levels in rabbits bearing the VX₂ carcinoma intra-abdominally did not vary significantly from those of nontumor-bearing rabbits. The differences in the serum calcium levels in rabbits bearing the VX₂ carcinoma at intramuscular and intra-abdominal implant sites may be related to different extents of metabolism by the lung and by the liver of prostaglandin E₂ or other cyclooxygense products of polyenoic fatty acids produced by the tumor.