Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion

Abstract Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells. HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.     

Recruitment of the Rhesus soleus and medial gastrocnemius before, during and after spaceflight

Electromyograms were recorded from the soleus and medial gastrocnemius muscles and tendon force from the medial gastrocnemius muscle of 2 juvenile Rhesus monkeys before, during and after Cosmos flight 2229 and of ground control animals. Recording sessions were made while the Rhesus were performing a foot pedal motor task. Preflight testing indicated normal patterns of recruitment between the soleus and medial gastrocnemius, i.e. a higher level of recruitment of the soleus compared to the medial gastrocnemius during the task. Recording began two days into the spaceflight and showed that the media gastrocnemius was recruited preferentially over the soleus. This observation persisted throughout the flight and for the 2 week period of postflight testing. These data indicate a significant change in the relative recruitment of slow and fast extensor muscles under microgravity conditions. The appearance of clonic-like activity in one muscle of each Rhesus during flight further suggests a reorganization in the neuromotor system in a microgravity environment.     

Fatty Acyl-CoA Inhibits 1-Alkyl-sn-Glycero-3-Phosphate Acetyltransferase in Microsomes of Immature Rabbit Cerebral Cortex: Control of the First Committed Step in the De Novo Pathway of Platelet-Activating Factor Synthesis

Abstract: Microsomal fractions of cerebral cortices of 15-day-old rabbits were used to study the 1-alkyl- sn -glycero-3-phosphate (AGP) acetyltransferase that generates 1-alkyl-2-acetyl- sn -glycero-3-phosphate in the de novo path of platelet-activating factor synthesis. The AGP acetyltransferase activity was inhibited by small concentrations of medium-long chain fatty acyl-CoA thioesters. In contrast, the AGP acyltransferase used oleoyl-CoA as substrate and was not inhibited by the presence of acetyl-CoA in high molar excess. The inhibition of AGP acetyltransferase was seen at concentrations of oleoyl-CoA as low as 0.5 µ M using 12.5 µ M AGP and 200 µ M acetyl-CoA. The inhibition by oleoyl-CoA was noncompetitive for the acetyl-CoA substrate. However, there was evidence that the oleoyl-CoA was competing with AGP in the acetyltransferase reaction, as the inhibition was lessened by increasing the AGP substrate concentration. Several acyl-CoA thioesters were effective as inhibitors of the AGP acetyltransferase, including oleoyl-, palmitoyl-, lauroyl-, and octanoyl-CoA. Propionyl- and butyryl-CoA were less effective as inhibitors, and propionyl-CoA was found to be a competitive inhibitor for acetyl-CoA. We have noted earlier that MgATP is an effective inhibitor of the AGP acetyltransferase and here we show that the inhibition by oleoyl-CoA can be increased by the presence of 0.1 m M MgATP. In brain ischemia, a decline in ATP levels would likely lead to a corresponding fall in acyl-CoA concentrations, thereby relieving the inhibition of AGP acetyltransferase and permitting the flow of AGP into the de novo pathway of platelet-activating factor synthesis.     

Adenosine effects upon insulin action on lipolysis and glucose transport in human adipocytes

The dose response effect of a new adenosine analogue, GR 79236 (N-[1S trans-2-hydroxycyclopentyl] adenosine) upon insulin sensitivity was examined in human adipocytes. The influence of adenosine upon insulin sensitivity for suppression of lipolysis and stimulation of glucose transport was examined. Removal of adenosine by use of adenosine deaminase stimulated lipolysis to the same extent as did 10^–9 M noradrenaline. GR79236 brought about dose dependent inhibition of lipolysis with half-maximal effect at 11.3±7.8×10^–9 M. When lipolysis was stimulated by noradrenaline alone the subsequent inhibition of lipolysis brought about by GR79236 was significantly greater than that of insulin. To examine adenosine effects on the insulin signalling pathway separately from those on lipolysis, the insulin sensitivity of glucose transport was examined. Removal of adenosine brought about a small but significant increase in the concentration of insulin required for half-maximal stimulation of glucose transport. Adenosine agonists offer promise as new agents for the modulation of metabolism in diabetes and other states of insulin resistance.     

Bio-oxidation of iron using Thiobacillus ferrooxidans

The effects of pH, ferrous and ferric ion concentrations on iron oxidation by Thiobacillus ferrooxidans were examined. The initial temperature and bacterial concentration were maintained at 37°C and 2±1×104cells/ml, respectively. The iron oxidation rate increased with increased initial ferrous iron concentration to 4g/l and thereafter decreased. The presence of iron(III) showed a negative effect on the bacterial iron oxidation rate. The increase of pH also showed an increase in the oxidation rate up to pH 1.75. The oxidation rate followed first order kinetics for the parameters studied. A rate equation has been developed.     

Long-Term Drug Treatment of Obesity in a Private Practice Setting

ATKINSON, RICHARD L, ROY C BLANK, DONALD SCHUMACHER, NIKHIL V DHURANDHAR, DOUGLAS L RITCH. Long-term drug treatment of obesity in a private practice setting. This study evaluated the long-term efficacy and safety of the combination of phentermine and fenfluramine for the treatment of obesity in a private practice setting. A total of 1388 consecutive, qualified patients presenting to a private general internal medicine practice in Charlotte, NC, were enrolled with eligibility criteria including: age 18 years to 60 years, 20% over “desirable” bodyweight or body mass index <27, no serious medical or psychiatric disease, and no contraindications to drug therapy. Patients were instructed in diet, exercise, and behavior modification techniques and received phentermine (15 mg/day to 30 mg/day) and fenfluramine (20 mg/day to 60 mg/day) continuously for over 3 years. Average duration of treatment was 15. 9 months, and average weight loss at the last visit was 11. 6 kg, or 11. 7% of initial bodyweight. For patients completing 1 year of drug treatment, mean weight loss was 16. 5 kg, or 16% of initial weight. Weight loss persisted for 2 years, but partial regain was seen at 3 years. The dropout rates were 18% at 6 months, 39% at 1 year, 68% at 2 years, and 78% at 3 years. At 1 year, blood pressure of hypertensive patients fell from 151/95 mm Hg to 127/78 mm Hg, and serum cholesterol and triglycerides of hyperlipidemic patients fell by 0. 750 mmol/L (29 mg/dL) and 0. 937 mmol/L (83 mg/dL), respectively. Adverse events were modest. We conclude that, in a private practice setting, long-term treatment of obesity with the combination of phentermine, fenfluramine, and a weight maintenance program is generally safe and effective. More research is needed to determine efficacy and safety for longer than 3 years.     

Differential Inhibition by Allylsulfide of Nitrification and Methane Oxidation in Freshwater Sediment

Addition of nitrapyrin, allylthiourea, C(inf2)H(inf2), and CH(inf3)F to freshwater sediment slurries inhibited CH(inf4) oxidation and nitrification to similar extents. Dicyandiamide and allylsulfide were less inhibitory for CH(inf4) oxidation than for nitrification. Allylsulfide was the most potent inhibitor of nitrification, and the estimated 50% inhibitory concentrations for this process and CH(inf4) oxidation were 0.2 and 121 (mu)M, respectively. At a concentration of 2 (mu)M allylsulfide, growth and CH(inf4) oxidation activity of Methylosinus trichosporium OB3b were not inhibited. Allylsulfide at 200 (mu)M inhibited the growth of M. trichosporium by approximately 50% but did not inhibit CH(inf4) oxidation activity. Nitrite production by cells of M. trichosporium was not significantly affected by allylsulfide, except at a concentration of 2 mM, when growth and CH(inf4) oxidation were also inhibited by about 50%. Methane monooxygenase activity present in soluble fractions of M. trichosporium was not inhibited significantly by allylsulfide at either 200 (mu)M or 2 mM. These results suggest that the partial inhibition of CH(inf4) oxidation in sediment slurries by high allylsulfide concentrations may be caused by an inhibition of the growth of methanotrophs rather than an inhibition of methane monooxygenase activity specifically. We conclude that allylsulfide is a promising tool for the study of interactions of methanotrophs and nitrifiers in N cycling and CH(inf4) turnover in natural systems.     

Callus Cultures from Zygotic Embryos of Costus speciosus and Their Morphogenetic Responses

Soft, nodular and hard types of calli were initiated on mature zygotic embryo explants of two tetraploid clones of Costus speciosus, of which, only the hard calli were amenable to morphogenetic responses. The two clones differed in their growth regulator requirements both for the initiation of calli and for shoot regeneration. De novo formation of both shoot bud meristems and somatic embryoids were observed. Latter were encased partially or fUlly by coleoptilar sheath. Embryoids could be isolated as discrete units. On maturity, a stock like appendage developed from the base and finally embryoids got detached from the subtending tissue. Both shoot-bud meristems and somatic embryoids developed into complete plantlets, the former upon sequential transfer of calli on Schenk and Hildebrandt’s (SH) basal medium containing lower levels of growth hormones, while the latter only on basal medium. These culture regenerants were subsequently transferred to the field. The morphogenetic behaviour of these two tetraploid clones reflects their marked genotypic difference inspite of their same ploidy status.     

Regulation by adenosine of the vasopressin-sensitive adenylate cyclase in pig-kidney cells (LLC-PK1L) grown in defined media

LLC-PK1L cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC-PK1L cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-L-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added. The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated.