Chemostat studies on the regulation of glucose metabolism in Pseudomonas aeruginosa by citrate

The effect of the relative concentrations of citrate and glucose on the regulation of key enzymes of the direct oxidative, phosphorylative, Entner-Doudoroff and pentose-cycle pathways of glucose metabolism in Pseudomonas aeruginosa has been investigated in continuous culture under conditions of NH(4) (+)-limitation. For comparison isocitrate dehydrogenase and aconitase were also assayed. Measurements were made for steady-state and transient conditions and the effect of growth rate was also studied. When cells grew on 75mm-citrate the glucose concentration had to attain 6-8mm before significant induction of enzymes of glucose metabolism occurred; the specific activities increased further as the result of both raising the glucose concentration to 30mm and then subsequently lowering the citrate to 60mm and then to 45mm. The specific activities of the glucose enzymes increased immediately during the transient period between the steady states characteristic of growth on 6mm- and 8mm-glucose, the increase continuing for about two doubling times. The converse experiment of adding increasing citrate concentrations to 45mm-glucose medium revealed an immediate induction of the citrate-transport system, oxidation of citrate following the increase in citrate concentration up to 8mm. Between 8mm- and 16mm-citrate a marked repression of gluconate, glucose 6-phosphate and 6-phosphogluconate dehydrogenases and the Entner-Doudoroff enzymes occurred. Increased growth rate in citrate medium resulted in decreased specific activities of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase. Increased growth rate in citrate-glucose medium gave decreased specific activities of isocitrate dehydrogenase and aconitase whereas the activities of some of the glucose enzymes decreased initially but then increased at the highest growth rate (0.5h(-1)), at which a marked increase in glucose utilization occurred. These observations accord with the regulation of glucose enzymes by induction with glucose or its metabolites and repression by citrate or its metabolic products.     

The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii

1. The enzymes beta-ketothiolase, acetoacetyl-CoA reductase, acetoacetate-succinate CoA-transferase (;thiophorase') and d(-)-3-hydroxybutyrate dehydrogenase have been partially purified from crude extracts of glucose-grown nitrogen-fixing batch cultures of Azotobacter beijerinckii. The condensation of acetyl-CoA to acetoacetyl-CoA catalysed by beta-ketothiolase is inhibited by CoASH, and the reverse reaction is inhibited by acetoacetyl-CoA. Acetoacetyl-CoA reductase has K(m) for acetoacetyl-CoA of 1.8mum and is inhibited by acetoacetyl-CoA above 10mum. The enzyme utilizes either NADH or NADPH as electron donor. The second enzyme of poly-beta-hydroxybutyrate degradation, d(-)-3-hydroxybutyrate dehydrogenase, is NAD(+)-specific and is inhibited by NADH, pyruvate and alpha-oxoglutarate. CoA transferase is inhibited by acetoacetate, the product of hydroxybutyrate oxidation. In continuous cultures poly-beta-hydroxybutyrate biosynthesis ceased on relaxation of oxygen-limitation and the rates in situ of oxygen consumption and carbon dioxide evolution of such cultures increased without a concomitant increase in glucose uptake. 2. On the basis of these and other findings a cyclic mechanism for the biosynthesis and degradation of poly-beta-hydroxybutyrate is proposed, together with a regulatory scheme suggesting that poly-beta-hydroxybutyrate metabolism is controlled by the redox state of the cell and the availability of CoASH, pyruvate and alpha-oxoglutarate. beta-Ketothiolase plays a key role in the regulatory process. Similarities to the pathways of poly-beta-hydroxybutyrate biosynthesis and degradation in Hydrogenomonas are discussed.     

Sutureless microvascular anastomoses by a biodegradable laser-activated solid protein solder.

A new sutureless technique to successfully anastomose the abdominal aorta of rats (1.3 mm in diameter) by using a fully biodegradable, laser-activated protein solder is presented. A total of 90 rats were divided into two groups randomly. In group one, the anastomoses were performed by using conventional microsuturing technique, whereas in group two, the anastomoses were performed by using a new laser welding technique. In addition, each of the two groups were divided into five subgroups and evaluated at different follow-up periods (10 minutes, 1 hour, 1 day, 1 week, and 6 weeks). At these intervals, the anastomoses were evaluated for patency and tensile strength. Three anastomoses in each subgroup were processed for light and electron microscopy. All anastomoses were found to be patent. The mean clamp time of the anastomoses performed with conventional suturing was 20.6 minutes compared with 7.2 minutes for the laser-activated welded anastomoses (p < 0.001). The strain measurements showed a stronger mechanical bond of the sutured anastomoses in the initial phase. However, at 6 weeks the tensile strength of the laser-welded anastomoses was higher compared with the conventional suture technique. Histologic evaluations revealed a near complete resorption of the solder after 6 weeks. The junction site of the vessel ends cannot be determined on the luminal side of the artery. In conclusion, a resorbable protein used as a solder, activated by a diode laser, can provide a reliable, safe, and rapid arterial anastomosis, which could be performed by any microsurgeon faster than conventional suturing after a short learning curve.