The active transport of 2-keto-D-gluconate in vesicles prepared from Pseudomonas purida.

The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax. of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1. The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax. In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol. 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively. The carrier system for 2KGA is repressed in vesicles from cells grown on succinate. Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors. Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx. 140 mV. The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol. Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p.     

Histamine H2 receptors on chondrocytes derived from human, canine and bovine articular cartilage.

Histamine (1-100 microM) induced a concentration-dependent increase in intracellular cyclic AMP in monolayer cultures of human, canine and foetal-bovine articular chondrocytes. The dose-response curve for histamine in each culture was progressively displaced to the right with increasing concentrations of cimetidine, an H2-receptor antagonist. The histamine-induced cyclic AMP elevation in human articular chondrocytes was also significantly decreased by ranitidine, another H2 antagonist, but not by the H1 antagonists mepyramine and chlorpheniramine. These findings indicate that histamine activates chondrocyte adenylate cyclase through an H2 receptor. The cyclic AMP response of human chondrocytes to histamine was many times greater than that measured for synovial fibroblasts under similar conditions. Such findings suggest that mast-cell-chondrocyte interactions in vivo may contribute to changed chondrocyte metabolism in joint disease.     

Chemosensitivity of crayfish slowly adapting stretch receptors to nicotine

It has recently been demonstrated that slowly adapting stretch receptors (SASRs) in the airways of the dog respond directly to nicotine (Federation Proc. 43: 318, 1984). The purpose of the present experiment was to investigate this chemical effect on an isolated stretch receptor. The crayfish muscle receptor organ was chosen, since crayfish muscle is reported to be insensitive to nicotine or acetylcholine and therefore permits the testing of any direct chemical effect of nicotine on the muscle stretch receptors. The tail was removed and pinned out in a tissue bath, and a stretch receptor organ was surgically isolated. Single-unit SASR extracellular nerve recordings were made while simultaneously measuring tension in the tail. Drugs were prepared in Van Harreveld's solution and administered into the bath kept at 18 degrees C. When resting muscle tension was essentially reduced to zero by cutting both ends of the receptor organ muscle, nicotine (0.07 microM) added to the bath increased receptor activity fourfold. This response was abolished by treatment with hexamethonium (690 microM). In a second group of animals in which the muscle was left intact, nicotine was shown to significantly increase receptor sensitivity to step changes in muscle tension. Once again hexamethonium blocked the response to nicotine. These results demonstrate that the sensitivity of mechanoreceptor can be altered by chemical interaction with nicotinic receptors, which dramatically alter sensory receptor activity.     

Mode of neural control mediating rat tail vasodilation during heating

The purpose of this investigation was to delineate the mode of efferent neural control mediating rat tail vasodilation during body heating. Tail blood flow (venous occlusion plethysmography), tail skin temperature over the ventral vascular bundle, and arterial pressure were measured in Sprague-Dawley rats anesthetized with pentobarbital sodium (45 mg/kg). Three protocols were followed: anesthesia of the lumbar sympathetic chain, bilateral lumbar sympathectomy, and sympathetic nerve stimulation during varying degrees of alpha-adrenergic receptor blockade. Mean tail blood flow and tail vascular conductance (TVC) during body heating were 40.3 +/- 8.7 ml X 100 ml-1 X min-1 and 39.2 +/- 9.2 ml X 100 ml-1 X min-1 X 100 mmHg-1, respectively. Interruption of sympathetic nerve activity by sympathetic nerve anesthetization or sympathectomy during heat stress caused a nonsignificant increase in TVC to 112.7 +/- 1.8 and 121.12 +/- 6.3%, respectively, of the values achieved with body heating. Sympathectomy performed in normothermic animals that had recovered from prior heating caused an increase in TVC to 128.4 +/- 14.0% of the levels achieved during the previous heating period. In addition, sympathetic nerve stimulation after complete alpha-adrenergic receptor blockade failed to produce a vasodilation [control TVC = 10.2 +/- 3.9 vs. TVC during nerve stimulation = 10.4 +/- 3.9 (P greater than 0.05)]. It is concluded that the increase in TVC during body heating occurs solely via a reduction in vasoconstrictor nerve activity.     

Osmotic reflection coefficient for total plasma protein in lung microvessels

The osmotic reflection coefficient (sigma) for total plasma proteins was estimated in 11 isolated blood-perfused canine lungs. Sigma's were determined by first measuring the capillary filtration coefficient (Kf,C in ml X min-1 X 100g-1 X cmH2O-1) using increased hydrostatic pressures and time 0 extrapolation of the slope of the weight gain curve. Kf,C averaged 0.19 +/- 0.05 (mean +/- SD) for 14 separate determinations in the 11 lungs. Following a Kf,C determination, the isogravimetric capillary pressure (Pc,i) was determined and averaged 9.9 +/- 0.5 cmH2O for all controls reported in this study. Then the blood colloids in the perfusate were either diluted or concentrated. The lung either gained or lost weight, respectively, and an initial slope of the weight gain curve (delta W/delta t)0 was estimated. The change in plasma protein colloid osmotic pressure (delta IIP) was measured using a membrane osmometer. The measured delta IIP was related to the effective colloid osmotic pressure (delta IIM) by delta IIM = (delta W/delta t)0/Kf,C = sigma delta IIP. Using this relationship, sigma averaged 0.65 +/- 0.06, and the least-squares linear regression equation relating Pc,i and the measured IIP was Pc,i = -3.1 + 0.67 IIP. The mean estimate of sigma (0.65) for total plasma proteins is similar to that reported for dog lung using lymphatic protein flux analyses, although lower than estimates made in skeletal muscle using the present methods (approximately 0.95).     

The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.     

Distribution and evolutionary significance of mitochondrial plasmids in Neurospora spp.

The lethal and mutagenic effects of various mutagens on Neisseria gonorrhoeae were investigated. Lethality studies demonstrated that N. gonorrhoeae was relatively sensitive to ethyl methanesulfonate, UV light, and methyl methanesulfonate. Although N. gonorrhoeae was readily mutated by ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine for the three genetic markers assayed, no increase in the mutation frequency was observed for any of the selective markers after UV irradiation or methyl methanesulfonate treatment. These results suggest that N. gonorrhoeae lacks an error-prone repair mechanism.     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Heterogeneity of macrophage populations in human lymphoid tissue and peripheral blood

Human lymphoid tissue and peripheral blood leukocytes were stained with six monoclonal antibodies directed against monocyte/macrophage populations. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages. The results show that there exists considerable heterogeneity of tissue macrophages based on the expression of surface and/or cytoplasmic antigens; furthermore, the distribution of cells bearing particular antigenic determinants is associated with distinct regions in normal lymphoid tissue. Double staining methods demonstrated that these antibodies bind to different, as well as to identical, macrophage populations. OKM-1 antibody binds predominantly to sinus histiocytes and tingible body macrophages. The Leu M-1 reagent stains interdigitating reticulum cells, while the KiM-4 antibody labels follicular dendritic cells. Leu M-3 antibody identifies cells predominantly in the germinal center, and histiocytes lining the sinuses. Both CM-1 and BRL-M.1 appear to stain tissue macrophages distributed throughout the lymphoid tissue.     

Mast cell products stimulate collagenase and prostaglandin E production by cultures of adherent rheumatoid synovial cells

Mast cells were purified from histologically-confirmed dog mastocytomas and extracted for whole mast cell products (MCP). When added to cultures of human adherent rheumatoid synovial cells MCP induced a 50-400 fold increase in prostaglandin E synthesis and a 10-50 fold stimulation of collagenase production. The mast cell stimulatory factor has not been identified and was not due to histamine, heparin or prostaglandin E. These results indicate a novel way in which mast cells might interact with synovial cells to promote the production of inflammatory mediators and proteolytic enzymes which might contribute to connective tissue degradation.