All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis

Glutamine synthetases (GS) are ubiquitous enzymes that play a central role in every cell's nitrogen metabolism. We have investigated the expression and activity of all four genomic Mycobacterium tuberculosis GS - GlnA1, GlnA2, GlnA3 and GlnA4 - and four enzymes regulating GS activity and/or nitrogen and glutamate metabolism - adenylyl transferase (GlnE), gamma-glutamylcysteine synthase (GshA), UDP-N-acetylmuramoylalanine-D-glutamate ligase (MurD) and glutamate racemase (MurI). All eight genes are located in multigene operons except for glnA1, and all are transcribed in M. tuberculosis; however, some are not translated or translated at such low levels that the enzymes escape detection. Of the four GS, only GlnA1 can be detected. Each of the eight genes, as well as the glnA1-glnE-glnA2 cluster, was expressed separately in Mycobacterium smegmatis, and its gene product was characterized and assayed for enzymatic activity by analysing the reaction products. In M. smegmatis, all four recombinant-overexpressed GS are multimeric enzymes exhibiting GS activity. Whereas GlnA1, GlnA3 and GlnA4 catalyse the synthesis of L-glutamine, GlnA2 catalyses the synthesis of D-glutamine and D-isoglutamine. The generation of mutants in M. tuberculosis of the four glnA genes, murD and murI demonstrated that all of these genes except glnA1 are nonessential for in vitro growth. L-methionine-S,R-sulphoximine (MSO), previously demonstrated to inhibit M. tuberculosis growth in vitro and in vivo, strongly inhibited all four GS enzymes; hence, the design of MSO analogues with an improved therapeutic to toxic ratio remains a promising strategy for the development of novel anti-M. tuberculosis drugs.     

A logical circuit for the regulation of fission yeast growth modes

Growth of fission yeast at the ends of its cylindrical cells switches from a monopolar to a bipolar mode, before it ceases during mitosis and cell division. Here we assume that these growth modes correspond to three stable states of an underlying regulatory circuit, which is a relatively simple and to a large degree autonomous subsystem of an otherwise complex cellular control system. We develop a switch-like logical circuit based on three elements defined as binary variables. Effects of circuit variables on each other are expressed in terms of logical operations. We analyse this circuit for its behavior ("phenotypes") after removing single or multiple operations ("mutants"). Known fission yeast polarity mutants such as those defective in the switch to bipolar growth can be classified based on these predicted 'phenotypes'. Differences in growth patterns between daughter cells in different bipolar growth mutants are also predicted by the circuit model. The model presented here should provide a useful framework to guide future experiments into mechanisms of cellular polarity. This paper illustrates the usefulness of simple logical circuits to describe and dissect features of complex regulatory processes such as the fission yeast growth patterns in both wild type and mutant cells.     

Mechanistic studies on a novel, highly potent gold-phosphole inhibitor of human glutathione reductase

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.     

Modeling nucleotide evolution at the mesoscale: the phylogeny of the neotropical pitvipers of the Porthidium group (viperidae: crotalinae)

We analyzed the phylogeny of the Neotropical pitvipers within the Porthidium group (including intra-specific through inter-generic relationships) using 1.4 kb of DNA sequences from two mitochondrial protein-coding genes (ND4 and cyt-b). We investigated how Bayesian Markov chain Monte-Carlo (MCMC) phylogenetic hypotheses based on this 'mesoscale' dataset were affected by analysis under various complex models of nucleotide evolution that partition models across the dataset. We develop an approach, employing three statistics (Akaike weights, Bayes factors, and relative Bayes factors), for examining the performance of complex models in order to identify the best-fit model for data analysis. Our results suggest that: (1) model choice may have important practical effects on phylogenetic conclusions even for mesoscale datasets, (2) the use of a complex partitioned model did not produce widespread increases or decreases in nodal posterior probability support, and (3) most differences in resolution resulting from model choice were concentrated at deeper nodes. Our phylogenetic estimates of relationships among members of the Porthidium group (genera: Atropoides, Cerrophidion, and Porthidium) resolve the monophyly of the three genera. Bayesian MCMC results suggest that Cerrophidion and Porthidium form a clade that is the sister taxon to Atropoides. In addition to resolving the intra-specific relationships among a majority of Porthidium group taxa, our results highlight phylogeographic patterns across Middle and South America and suggest that each of the three genera may harbor undescribed species diversity.     

A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.     

Dendritic cells acquire tolerogenic properties at the site of sterile granulomatous inflammation

Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4(+) T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II(+) His 24(+) His 48(+) DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4(+) T cells. These results correlated with the increased levels of IL-10 and TGF-beta in culture supernatants and the sponge exudate.     

Budding, vesiculation and permeabilization of phospholipid membranes-evidence for a feasible physiologic role of beta2-glycoprotein I and pathogenic actions of anti-beta2-glycoprotein I antibodies

The in vivo physiologic role of beta2-glycoprotein I (beta2GPI) is presumed to be related to its interactions with negatively charged phospholipid membranes. Increased quantities of procoagulant microparticles derived by the vesiculation of blood cells have been detected in patients with antiphospholipid syndrome (APS) frequently associated with antibodies against beta2GPI (anti-beta2GPI). We investigated the influence of beta2GPI and anti-beta2GPI on giant phospholipid vesicles (GPVs). GPVs composed of phosphatidylserine and phosphatidylcholine were formed in an aqueous medium and individually transferred to a compartment containing either beta2GPI, anti-beta2GPI, or beta2GPI along with anti-beta2GPI. Shape changes of a single GPV were observed by a phase contrast microscope. Most GPVs transferred to the solution containing only beta2GPI budded moderately. Upon the transfer of GPVs to the solution containing beta2GPI and anti-beta2GPI either from patient with APS or mouse monoclonal anti-beta2GPI Cof-22, the budding was much more pronounced, generating also daughter vesicles. No such effects were seen when GPV was transferred to the solution containing anti-beta2GPI without beta2GPI. Our results suggest a significant physiologic role of beta2GPI in the budding of phospholipid membranes, which may be explained by the insertion of the C-terminal loop of beta2GPI into membranes, thus increasing the surface of the outer layer of a phospholipid bilayer. Anti-beta2GPI, recognizing domains I to IV of beta2GPI, enhanced the budding and vesiculation of GPVs in the presence of beta2GPI. This might be a novel pathogenic mechanism of anti-beta2GPI, promoting in vivo the expression of proadhesive and procoagulant phospholipid surfaces in APS.     

Accumulation of N-acetyl-L-aspartate in the brain of the tremor rat, a mutant exhibiting absence-like seizure and spongiform degeneration in the central nervous system

The tremor rat is a mutant that exhibits absence-like seizure and spongiform degeneration in the CNS. By positional cloning, a genomic deletion was found within the critical region in which the aspartoacylase gene is located. Accordingly, no aspartoacylase expression was detected in any of the tissues examined, and abnormal accumulation of N-acetyl-L-aspartate (NAA) was shown in the mutant brain, in correlation with the severity of the vacuole formation. Therefore, the tremor rat may be regarded as a suitable animal model of human Canavan disease, characterized by spongy leukodystrophy that is caused by aspartoacylase deficiency. Interestingly, direct injection of NAA into normal rat cerebroventricle induced 4- to 10-Hz polyspikes or spikewave-like complexes in cortical and hippocampal EEG, concomitantly with behavior characterized by sudden immobility and staring. These results suggested that accumulated NAA in the CNS would induce neuroexcitation and neurodegeneration directly or indirectly.     

A relative high dose of vitamin E does not attenuate unweighting-induced oxidative stress and ubiquitination in rat skeletal muscle

We previously reported that intragastric administration of cysteine could be beneficial to prevent unweighting-induced ubiquitination and degradation of muscle protein in association with redox regulation [Ikemoto et al., Biol. Chem., 383 (2002), 715-721]. In this study, we investigated whether vitamin E, another potent antioxidative nutrient, also had beneficial effects on the muscle protein catabolism. However, daily intragastric supplementation of 1.5 or 15 mg/rat of alpha-tocopherol did not prevent weight loss of hindlimb skeletal muscle in tail-suspended rats. To elucidate the reason for the non-effectiveness of vitamin E, we further examined concentrations of oxidative stress markers, ubiquitination of muscle proteins and fragmentation of myosin heavy chain in gastrocnemius muscle of rats daily treated with 15 mg of alpha-tocopherol. Unexpectedly, vitamin E increased concentrations of glutathione disulfide and thiobarbituric acid-reactive substance and decreased glutathione level in the muscle, compared with those of vehicle treatment, indicating that vitamin E enhanced unweighting-induced oxidative stress in skeletal muscle. The vitamin E supplementation did not suppress the ubiquitination of muscle proteins and fragmentation of myosin heavy chain caused by tail-suspension. Our results suggest that supplementation of a relative high dose of vitamin E could not inhibit ubiquitin-dependent degradation of muscle protein in tail-suspended rats possibly due to its prooxidant action.     

Survival and blood flow evaluation of canine venous flaps

Using a canine model, we compared postoperative viability of saphenous venous flaps, cephalic venous flaps, and composite-tissue grafts without vascular connections. Of the saphenous flaps, 14 percent survived. Of the flaps based on the cephalic vein, 75 percent survived. Cephalic composite-tissue grafts were 13 percent successful. The presence of a more intricate venous plexus in a flap seems to increase its chances of success. Arterial injections of radioisotope-labeled microspheres were used to chart revascularization in cephalic flaps. These flaps demonstrated arterial blood flow by day 3, while the composite grafts showed no flow until day 7. Venous injections of microspheres distal to the flap were used to test vein-to-capillary blood flow. No significant entrapment of microspheres within the flaps occurred at any time, suggesting such flow to be inadequate.