Fructosebisphosphatase isoenzymes of the chemoautotroph Xanthobacter flavus.

Xanthobacter flavus employs two fructosebisphosphatase (FBPase)-sedoheptulosebisphosphatase (SBPase) enzymes. One of these is constitutively expressed and has a high FBPase-to-SBPase ratio. The alternative enzyme, which is encoded by cbbF, is induced during autotrophic growth. The cbbF gene was expressed in Escherichia coli, and the FBPase was purified to homogeneity. The purified enzyme has a specific FBPase activity of 114 mumol/min/mg of protein, a Michaelis constant for fructosebisphosphate of 3 microM, and a low FBPase-to-SBPase ratio. CbbF was activated by ATP and inhibited by Ca2+.     

Permeability of blood-tear barrier to fluorescein and albumin after application of platelet-activating factor to the eye of the guinea pig

One of the inflammatory responses of the eye to local application of platelet-activating factor (PAF) is oedema of the conjunctiva, caused by extravasation of plasma. Aim of the study was to investigate if fluorescein would leak from the blood into the tears together with plasma protein after application of PAF to the eye. Fluorescein was given intraperitoneally 30 min prior to application of 25 microl of 0.1% solution of PAF. Thirty min after PAF the tear film was collected by washing the surface of the eye with 25 microl of phosphate buffered saline (PBS). Fluorescein in eye washings and in plasma was measured by fluorophotometry and albumin by immunodiffusion. Both fluorescein and albumin appeared in a related fashion in tears, being absent in washings of placebo-treated control eyes. Extravasation of fluorescein can be used as a measure for plasma leakage in the conjunctiva with the advantage over the Evans Blue method that the former is a non-invasive method.     

Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors,olomoucine and isopentenyladenine

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(δ2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley-Liss, Inc.     

Human cytomegalovirus induces a cellular deoxyguanosine kinase, also interacting with Acyclovir

Abstract A cytosol deoxyguanosine kinase (dGK) is induced in either growing or human cytomegalovirus (HCMV, AD169)-infected human fibroblasts (HEF). Data obtained from polyacrylamide gel electrophoresis, heat inactivation and phosphorylation kinetic experiments proved that these dGKs are identical, but completely differ from HCMV-induced thymidine kinase (TK) or deoxycytidine kinase (dCK). In contrast to TK or dCK, only dGK interacts with Acyclovir ( K _i = 590 μ M). It is suggested that dGK is an important enzyme determining the antiviral activity of Acyclovir.     

Migration of leafhoppers (Homoptera: Auchenorrhyncha) into a new polder

Auchenorrhyncha of a new polder were sampled by different kinds of traps in four stations in new habitats, one station on the salt marsh and one just outside the polder. In the latter stations 20 species of Auchenorrhyncha were caught in numbers that declined during the four year study. The proportion of macropters of the common Streptanus sordidus was highest in the largest catches. In the polder proper 392 specimens of 13 species were caught, all had moved at least 1500 m, but some could be traced to origins at greater distances. The number of immigrants also declined during the study. The distribution of the leafhoppers over the trapping stations suggests that leafhoppers, although sometimes transported over great distances, as a rule quickly fall out once they are airborne. Probably only one species had founded a resident population in the new polder at the end of the study.     

Carbon metabolism of Frankia spp. in root nodules of Alnus glutinosa and Hippophaë rhamnoides

The occurrence and localization of enzymes involved in glycolysis, tricarboxylic acid cycle and glyoxylate cycle in root nodules of Alnus glutinosa (L.) Vill. and Hippophaë rhamnoides L. ssp. rhamnoides were studied. The following enzymes, catalyzing reversible steps in the glycolysis, were found in both the endophyte Frankia spp. and the plant cytosol of Alnus nodules: fructose-1,6-diphosphate aldolase, glyceralde-hyde-3-phosphate dehydrogenase, phosphoglycerate kinase and enolase. The enzymes catalyzing irreversible steps in glycolysis, viz. hexokinase and pyruvate kinase, were detectable only in the plant cytosol. Similar results were obtained with nodule homogenates of Hippophaë. This indicates the absence of a complete glycolysis in the endophyte. Vesicle clusters of the nodule endophyte of Alnus contained various dehydrogenases of the tricarboxylic acid cycle and showed activity of glutamate oxaloacetate transaminase. Respiration studies showed that vesicle clusters take up oxygen when supplied with NAD, glutamate and malate together. No oxygen uptake was found when any of these compounds was omitted. Vesicle clusters from both Alnus and Hippophaë nodules showed no detectable activity of the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Since these enzymes are known to be present in Frankia Avcll, when grown in a medium with Tween 80 as carbon source, it is suggested that the glyoxylate cycle enzymes are repressed in the root-nodule symbioses.     

Inhibition of urea-cycle activity by high concentrations of alanine.

1. Conditions are described in which high intracellular alanine concentrations inhibit urea-cycle flux in isolated hepatocytes. 2. Inhibition of urea-cycle flux by added alanine is DL-cycloserine-insensitive and is accompanied by an increase in intracellular citrulline and a decrease in ornithine. 3. Argininosuccinate synthetase (EC 6.3.4.5) activity in rat liver cytosol is inhibited by alanine in a competitive manner with respect to citrulline. It is concluded that this effects is the primary cause of inhibition of urea-cycle flux by alanine.     

The increase in activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in skeletal muscles of rats after subcutaneous administration of N,N′-dimethyl-para-phenylenediamine

Summary After subcutaneous administration of N,N-dimethyl-para-phenylenediamine (DPPD) in rats, a myogenic myopathy was produced in the skeletal muscles. In this communication, the results of the application of various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases and biochemical techniques for the estimation of activities of oxidoreductases in the experimental skeletal muscles are presented. The most striking result was the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase which increased dramatically during the early phase of the muscle disease. The increase in activity of the pentose phosphate shunt enzymes was the first pathological alteration and was present as early as 8 h after a single injection of DPPD. Histochemical techniques for demonstration of activity of both enzymes are therefore highly suited for the detection of minor diseases and the early onset of major diseases of the neuromuscular system. Some glycolytic enzymes as well as some enzymes of the aerobic part of the metabolism showed an early decrease or increase in activity indicating a metabolic imbalance in the muscle fibres. There were more fibres with an intermediate pattern of the energy yielding enzymes in the experimental muscle specimens then in specimens from the control groups. The activity of the catabolic hydrolytic enzymes was strongly increased in pathological muscles. The aerobic muscles were more vulnerable to DPPD than the anaerobic muscles.     

Biochemical investigation to the reliability of the histochemical demonstration of microsomal arylsulphatase activity in cryostat sections

Summary Polyacrylamide gel-electrophoresis was performed with an extract from cultivated skin fibroblasts. Arylsulphatase activity is measured and visualised using the biochemical substrate dehydroepiandrosterone sulphate and the histochemical substrate 6-bromo-2-naphthyl sulphate respectively. The histochemical substrate was hydrolysed at Rf=0.49 and 0.58 while the biochemical substrate was hydrolysed only at 0.49. We conclude that two different microsomal arylsulphatases exist: a sulphatase able to hydrolyse steroid sulphatases (Rf=0.49) and one unable to hydrolyse steroid sulphatases (Rf=0.58). In consequence it is recommended to carry out an electrophoresis experiment after the histochemical investigation, in order to discriminate between these two types of sulphatase.     

Energy conservation during nitrate respiration in Paracoccus denitrificans

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the Y _ATP ^max values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate-and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source the average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively.H⁺/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate-and nitratelimited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate-and nitritelimitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.