Tn5 insertion mutants of Pseudomonas aeruginosa deficient in surface expression of ferripyochelin-binding protein.

Transposon (Tn5) insertion mutants were isolated in Pseudomonas aeruginosa PAO. These mutants were screened for expression of the ferripyochelin-binding protein with monoclonal antibody in a whole-cell immunoblot assay. Fourteen mutants were identified which did not express ferripyochelin-binding protein on the cell surface. These mutants did not take up 59Fe-labeled pyochelin and grew slowly in the presence of iron chelators.     

Sulfhydryl groups are essential for organic anion exchange in canine renal brush-border membranes

The effect of several sulfhydryl-modifying reagents (HgCl2, p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide) on the renal organic anion exchanger was studied. The transport of p-amino[3H]hippurate, a prototypic organic anion, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. HgCl2, PCMBS and N-ethylmaleimide inactivated p-aminohippurate transport with IC50 values of 38, 78 and 190 microM. The rate of p-aminohippurate inactivation by N-ethylmaleimide followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the N-ethylmaleimide concentration with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of N-ethylmaleimide inactivates one essential sulfhydryl group per active transport unit. Substrate (1 mM p-aminohippurate) affected the rate of the N-ethylmaleimide (1.3 mM) inactivation: the t1/2 values for inactivation in the presence and absence of p-aminohippurate were 7.4 and 3.7 min, respectively. The results demonstrate that there are essential sulfhydryl groups for organic anion transport in the brush-border membrane. Moreover, the ability of substrate to alter sulfhydryl reactivity suggests that the latter may play a dynamic role in the transport process.     

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.     

Lead toxicity and the hypothalamic-pituitary-testicular axis

Environmental exposure to toxic levels of lead occurs in a number of industries with potential adverse effects on the reproductive capacity of exposed men. Clinical and animal studies indicate that abnormalities of spermatogenesis result from toxic lead exposure, but the pathogenetic mechanisms involved have not been identified. In order to ascertain what reproductive abnormalities occur in experimental animals when exposed to low levels of lead, 52-day-old animals were treated with water containing 0.0% (control), 0.1%, or 0.3% lead acetate for 30 days prior to killing. Whole blood serum lead levels were below detection (less than 7 micrograms/dl) in the control animals, 34 +/- 3 micrograms/dl in the 0.1% group, and 60 +/- 4 micrograms/dl in the 0.3% group (P less than 0.001). Significant negative correlations between whole blood lead levels and serum and intratesticular testosterone values were found (r = 0.64, P less than 0.001 and r = 0.6, P less than 0.001, respectively). As the level of lead exposure increased, intratesticular sperm counts significantly decreased (r = 0.81, P less than 0.001). No significant changes in serum luteinizing hormone (LH) values were found, but sperm follicle-stimulating hormone (FSH) values were significantly suppressed (P less than 0.05) after lead treatment. There was a significant decrease in ventral prostate weight (P less than 0.05), but no differences in testicular or seminal vesicle weights. Our data indicate that dietary exposure to lead resulting in whole blood serum lead values considered acceptable in the workplace (less than or equal to 40 micrograms/dl) causes inhibition of testicular function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometry of monocytes of human blood

Morphometric methods have been used to analyse the ultrastructural characteristics of human blood monocytes in 20 normal adults. Data were obtained relating to whole cell, nucleus and mitochondria and, using a method not previously applied to blood cells, for the distribution of heterochromatin and euchromatin. The results provide a normal baseline against which monocyte changes in disease can be assessed.     

Release of Envelope Glycoprotein from Rabies Virions by a Nonionic Detergent

Treatment of rabies virus with the nonionic detergent Nonidet P-40 resulted in solubilization of viral lipids and in a preferential release of the envelope glycoprotein. The other viral proteins and the viral ribonucleic acid remained associated in "core" particles sedimenting at a rate similar to that of intact virions. After fractionation of treated virus by velocity centrifugation in a sucrose density gradient, the amount of residual glycoprotein recovered in the "core" particle fraction and the extent of contamination of the glycoprotein fraction by other viral components were dependent on the ratio of detergent to viral protein used.     

Fluorescent antibody method Conjugation Of Fluoresceinisothiocyanate With Immune ?-Globulin

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Some Medical Problems of Chronic Hemodialysis

Chronic intermittent hemodialysis may relieve some medical problems of terminal uremia (for example, azotemia, acidosis, hypertension, neuro-muscular disorders, bleeding, pericarditis) to such a degree that many patients are able to resume their normal activity. There remain, however, problems which are not readily changed by hemodialysis (anemia, peripheral neuropathy, pruritus, sexual impotence, renal osteodystrophy). These, together with medical problems possibly caused by hemodialysis (for example, osmotic disequilibrium, errors in dialysate composition, hepatitis, hemosiderosis, isoimmunization from blood transfusions, shunt problems and psychological problems of dependency upon the artificial kidney) represent a limitation of the present type of hemodialysis therapy.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.