Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.     

Superoxide dismutase is upregulated in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> following protoporphyrin-mediated photodynamic inactivation and does not directly influence the response to photodynamic treatment

Background  

Staphylococcus aureus, a major human pathogen causes a wide range of disease syndromes. The most dangerous are methicillin-resistant S. aureus (MRSA) strains, resistant not only to all β-lactam antibiotics but also to other antimicrobials. An alarming increase in antibiotic resistance spreading among pathogenic bacteria inclines to search for alternative therapeutic options, for which resistance can not be developed easily. Among others, photodynamic inactivation (PDI) of S. aureus is a promising option. Photodynamic inactivation is based on a concept that a non toxic chemical, called a photosensitizer upon excitation with light of an appropriate wavelength is activated. As a consequence singlet oxygen and other reactive oxygen species (e.g. superoxide anion) are produced, which are responsible for the cytotoxic effect towards bacterial cells. As strain-dependence in photodynamic inactivation of S. aureus was observed, determination of the molecular marker(s) underlying the mechanism of the bacterial response to PDI treatment would be of great clinical importance. We examined the role of superoxide dismutases (Sod) in photodynamic inactivation of S. aureus as enzymes responsible for oxidative stress resistance.     

Synthesis and structure-activity relationship of 2-(aminoalkyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives: a novel series of 5-HT(2A/2C) receptor antagonists. Part 2.

Following the programme started at Janssen Research Foundation searching for 5-HT(2A/2C) antagonists, we now report on the synthesis of a series of substituted 2-(Dimethylaminomethyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives. The 5-HT(2A), 5-HT(2C) and H(1) receptor affinities as well as the mCPP antagonistic activity of the compounds synthesised is described.     

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of indole-3-acetyl-myo-inositol in immature endosperm of Zea mays

1- O -(indole-3-acetyl)- β - d -glucose: myo -inositol indoleacetyl transferase (IA- myo -inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1- O -(indole-3-acetyl)- β - d -glucose to myo -inositol to form IA- myo- inositol and glucose. IA- myo -inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion-exchange, adsorption on hydroxylapatite, affinity chromatography on ConA-Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their R _f on 8% polyacrylamide gel. The preparation of R _f 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of R _f 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS-PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo- or heterodimeric form (110–130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino-acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase-like (SCPL) acyltransferase family.