Application of FT-IR spectroscopy for control of the medium composition during the biodegradation of nitro aromatic compounds

Previous studies showed that cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation of nitro aromatic compounds by specific consortium of bacteria upon their growth. For further development of the approach for contaminated soil remediation it was necessary to evaluate the qualitative and/or quantitative composition of different origin CLE and their relevance on the growth of explosives-degrading bacteria. Six CLE (different by species, cultivars and harvesting time) were tested and used as additives to the growth medium. It was shown that nitro aromatic compounds can be identified in the FT-IR absorption spectra by the characteristic band at 1,527 cm⁻¹, and in CLE by the characteristic band at 1,602 cm⁻¹. The intensity of the CLE band at 1,602 cm⁻¹ correlated with the concentration of total nitrogen (R ² = 0.87) and decreased upon the growth of bacteria. The content of nitrogen in CLE differed (0.22–1.00 vol.%) and significantly influenced the content of total carbohydrates (9.50–16.00% DW) and lipids [3.90–9.90% dry weight (DW)] accumulated in bacterial cells while the content of proteins was similar in all samples. Though this study showed quantitative differences in the composition of the studied CLE and the response of bacterial cells to the composition of the growth media, and proved the potential of this additive for remediation of contaminated soil. It was shown that analysis of CLE and monitoring of the conversion of nitro aromatic compounds can be investigated by FT-IR spectroscopy as well as by conventional chemical methods.     

Immune interactions in malaria co-infections with other endemic infectious diseases: implications for the development of improved disease interventions

Today targeted research efforts are in progress with the goal to develop vaccines, microbicides, new drugs and alternative treatments for some of the neglected infectious diseases (NIDs). Until now the world is far from having effective cures and/or prophylactic vaccines in place. People living in endemic areas generally are more skewed towards a Th2 profile (i.e. anti-inflammatory) that could greatly affect the induction of an inflammatory Th2 type response needed to combat many infectious microorganisms. Despite this, very little is today known about how co-infections with NID can affect the outcome of the different diseases and the possibilities for prophylactic vaccination and treatment. Thus, if we are to intervene successfully to eradicate infections or prevent immune pathology either by vaccination or other immune intervention therapies it will be crucial to understand how co-infections with different pathogens affect the adaptive immunity and the establishment of immunological memory The aim of this paper is to review what is known about co-infection with malaria and certain other pathogens.     

An enhanced miniaturized assay for antimicrobial prospecting

Bioprospecting for novel antimicrobials increasingly relies on extremely small samples unsuitable for conventional bulk extraction and assay. We developed a microtitre plate assay for minimal amounts of test materials which is rapid, extremely sensitive, allows time-course analysis and reduces false negatives. Developed for the analyses of antimicrobial sensitivity and resistance, the technique is appropriate for assays where source materials are scarce.     

Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a beta-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: (alpha/alpha)(5) for CS (chondroitin lyases AC) and beta-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located approximately 12 A from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.     

Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis

Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) methylates, in vitro, caffeoyl-CoA and 5-hydroxyferuloyl-CoA, two possible precursors in monolignol biosynthesis in vivo. To clarify the in vivo role of CCoAOMT in lignin biosynthesis, transgenic poplars with 10% residual CCoAOMT protein levels in the stem xylem were generated. Upon analysis of the xylem, the affected transgenic lines had a 12% reduced Klason lignin content, an 11% increased syringyl/guaiacyl ratio in the noncondensed lignin fraction, and an increase in lignin-attached p-hydroxybenzoate but otherwise a lignin composition similar to that of wild type. Stem xylem of the CCoAOMT-down-regulated lines had a pink-red coloration, which coincided with an enhanced fluorescence of mature vessel cell walls. The reduced production of CCoAOMT caused an accumulation of O(3)-beta-d-glucopyranosyl-caffeic acid, O(4)-beta-d-glucopyranosyl-vanillic acid, and O(4)-beta-d-glucopyranosyl-sinapic acid (GSA), as authenticated by (1)H NMR. Feeding experiments showed that O(3)-beta-d-glucopyranosyl-caffeic acid and GSA are storage or detoxification products of caffeic and sinapic acid, respectively. The observation that down-regulation of CCoAOMT decreases lignin amount whereas GSA accumulates to 10% of soluble phenolics indicates that endogenously produced sinapic acid is not a major precursor in syringyl lignin biosynthesis. Our in vivo results support the recently obtained in vitro enzymatic data that suggest that the route from caffeic acid to sinapic acid is not used for lignin biosynthesis.     

Prothrombin overexpressed in post-natal neurones requires blood factors for activation in the mouse brain

Thrombin is thought to mediate, through protease-activated receptors, both protective as well as cytotoxic effects. As thrombin receptors are expressed in the CNS, an important question arises as to whether the intact nervous system is able to generate thrombin by activation of its precursor prothrombin, derived endogenously or only upon extravasation following brain injury. To address this question, transgenic mice that express C-terminally haemagglutinin tagged human prothrombin in post-mitotic neurones were generated. In situ hybridization and immunohistochemical analysis showed abundant and widespread cerebral expression of the transgene. Amidolytic assays of brain homogenates and hippocampal slice cultures demonstrated that activation of transgenic prothrombin required added factors, such as snake venom or blood components. This strongly suggests that any possible action of thrombin in the adult CNS depends on blood-derived factors that activate prothrombin. Furthermore, the results are consistent with the idea that in the non-pathological situation an as yet unidentified ligand activates thrombin receptors in the nervous system.     

Male mice lacking three germ cell expressed genes are fertile

In recent years, much knowledge about the functions of defined genes in spermatogenesis has been gained by making use of mouse transgenic and gene knockout models. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp-2), proacrosin (Acr), histone H1.1 (H1.1), and histone H1t (H1t), have been generated and analyzed. Tnp-2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues and germ cells during the S-phase of the cell cycle. The histone gene H1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization, two lines of triple null mice (Tnp-2-/-/Acr-/-/H1.1-/- and Tnp-2-/-/Acr-/-/H1t-/-) were established. Both lines are fertile and show normal sperm parameters, which clearly demonstrate the functional redundancy of these proteins in male mouse fertility. However, sperm only deficient for Acr (Acr-/-) are able to compete significantly with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1.1-/- (70.7% vs. 29.3%) but not with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1t-/- (53.6% vs. 46.4%). These results are consistent with a model that suggests that some sperm proteins play a role during sperm competition.     

Phosphorothioate-stimulated uptake of short interfering RNA by human cells

The cellular delivery of short interfering RNA (siRNA) is a main hurdle in therapeutic drug development. Here, we describe that phosphorothioate (PTO)-derived oligonucleotides stimulate the physical cellular uptake of siRNA in trans in human cells. This is reflected by an apparent dose-dependent siRNA-mediated suppression of lamin A/C in primary human umbilical vein endothelial cells. The PTO-stimulated cellular uptake in trans is concentration dependent, length dependent, related to the phosphorothioate chemistry but not sequence specific. We provide experimental evidence to support a caveolin-mediated uptake mechanism. In sum, this work strongly suggests the exploration of PTOs as facilitators in the delivery of biologically active siRNA to mammalian cells.