Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn(2+), NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC(50) value of 80 nm. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.     

Augmented lipopolysaccharide-induced TNF-alpha production by peritoneal macrophages in type 2 diabetic mice is dependent on elevated glucose and requires p38 MAPK

Dysregulated inflammation is a complication of type 2 diabetes (T2D). In this study, we show that augmented LPS-induced TNF-alpha production by resident peritoneal macrophages (PerMphi) in type 2 diabetic (db/db) mice is dependent on elevated glucose and requires p38 MAPK. Intraperitoneal LPS administered to db/db and nondiabetic (db/+) mice induced 3- and 4-fold more TNF-alpha in the peritoneum and serum, respectively, of db/db mice as compared with db/+ mice. Examination of the TLR-4/MD2 complex and CD14 expression showed no difference between db/db and db/+ PerMphi. Ex vivo stimulation of PerMphi with LPS produced a similar 3-fold increase in TNF-alpha production in db/db PerMphi when compared with db/+ PerMphi. PerMphi isolated from db/+ mice incubated in high glucose (4 g/L) medium for 12 h produced nearly 2-fold more TNF-alpha in response to LPS than PerMphi incubated in normal glucose medium (1 g/L). LPS-dependent stimulation of PI3K activity, ERK1/2 activation, and p38 kinase activity was greater in PerMphi from db/db mice as compared with db/+ mice. Only inhibition of p38 kinase blocked LPS-induced TNF-alpha production in PerMphi from db/db mice. Taken together, these data indicate that augmented TNF-alpha production induced by LPS in macrophages during diabetes is due to hyperglycemia and increased LPS-dependent activation of p38 kinase.     

Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor

Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities. We show here that specific, but not all, glucocorticoid receptor isoforms induced apoptosis in human osteosarcoma U-2 OS bone cells. Whole human genome microarray analysis revealed that the majority of the glucocorticoid target genes were selectively regulated by specific glucocorticoid receptor isoforms. Real-time PCR experiments confirmed that proapoptotic enzymes necessary for cell death, granzyme A and caspase-6, were induced by specific glucocorticoid receptor isoforms. Chromatin immunoprecipitation assays further suggested that glucocorticoid receptor isoform-dependent induction of proapoptotic genes was likely due to selective coregulator recruitment and chromatin modification. Interestingly, the capabilities to transrepress proinflammatory genes were similar among glucocorticoid receptor isoforms. Together, these findings provide new evidence that translational glucocorticoid receptor isoforms can elicit distinct glucocorticoid responses and may be useful for the development of safe glucocorticoids with reduced side effects.     

Central Charged Residues in DIIIS4 Regulate Deactivation Gating in Skeletal Muscle Sodium Channels

Summary 1. Mutations in the S4 segment of domain III in the voltage gated skeletal muscle sodium channel hNa_V1.4 were constructed to test the roles of each charged residue in deactivation gating. Mutations comprised charge reversals at K1-R6, charge neutralization, and substitution at R4 and R5. 2. Charge-reversing mutations at R4 and R5 produced the greatest alteration of activation parameters compared to hNa_V1.4. Effects included depolarization of the conductance/voltage (g/V) curve, decreased valence and slowing of kinetics. 3. Reversal of charge at R2 to R4 hyperpolarized, and reversal at R5 or R6 depolarized the h _∞ curve. Most DIIIS4 mutations slowed inactivation from the open state. R4E slowed closed state fast inactivation and R5E inhibited its completion. 4. Deactivation from the open and/or inactivated state was prolonged in mutations reversing charge at R2 to R4 but accelerated by reversal of charge at R5 or R6. Effects were most pronounced at central charges R4 and R5. 5. Charge and structure each contribute to effects of mutations at R4 and R5 on channel gating. Effects of mutations on activation and deactivation at R4 and, to a lesser extent R5, were primarily owing to charge alteration, whereas effects on fast inactivation were charge independent.     

Complete inhibition of the Pdr5p multidrug efflux pump ATPase activity by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source

The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.     

High level accumulation of α-glucan in maize kernels by expressing the <Emphasis Type="Italic">gtfD</Emphasis> gene from <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Glucosyltransferases (GTFs, EC.2.4.1.5) are bacterial enzymes that catalyze the polymerization of glucose residues from sucrose, leading to the production of high molecular weight glucan with α-1,3 /α-1,6 linkages. Such glucans, with many potential food and industrial applications, do not normally exist in higher plants. We fused a mutant form of the gtfD gene from Sreptococcus mutans with the maize (Zea mays L.) chloroplastic Brittle 1 transit peptide for amyloplast targeting. This construct, driven by the ubiquitin promoter, was introduced into maize by Agrobacterium-mediated transformation. We developed a novel HPLC-based method that enabled us differentially to distinguish transgene glucan from other endogenous polysaccharides in maize kernels. Using this method, we screened over 100 transgenic plants for the presence of GTF-produced glucan whose content varied between 0.8 and 14% of dry weight in the mature transgenic seeds. The mature transgenic plants were indistinguishable from wildtype plants in growth rate and morphology. Furthermore, starch granule size in the transgenic maize kernel was unaffected by the accumulation of the foreign polysaccharide. Mutation in Sh2, which encodes a subunit of ADP-glucose pyrophosphorylase, had no effect on glucan accumulation caused by gtfD expression. Our results indicated that high levels of novel carbohydrate polymer can be accumulated in crop plants through transgene technology.     

Phase 0 clinical trials in cancer drug development: from FDA guidance to clinical practice

The Food and Drug Administration (FDA) recently introduced the Exploratory Investigational New Drug Guidance to expedite the clinical evaluation of new therapeutic and imaging agents. Early clinical studies performed under the auspices of this guidance, so-called "Phase 0" trials, have been initiated at the National Cancer Institute to integrate qualified pharmacodynamic biomarker assays into first-in-human cancer clinical trials of molecularly targeted agents. The goal of this integration is to perform molecular proof-of-concept investigations at the earliest stage of cancer drug development. Phase 0 trials do not offer any possibility of patient benefit; instead, intensive, real-time pharmacodynamic and pharmacokinetic analyses of patient tumor samples and/or surrogate tissues are performed to inform subsequent trials. Phase 0 studies do not replace formal Phase I drug safety testing and require a substantial investment of resources in assay development early on; however, they offer the promise of more rational selection of agents for further, large-scale development as well as the molecular identification of potential therapeutic failures early in the development process.     

Non-breeding season habitat quality mediates the strength of density-dependence for a migratory bird

Our understanding of when natural populations are regulated during their annual cycle is limited, particularly for migratory species. This information is needed for parametrizing models that can inform management and conservation. Here, we use 14 years of data on colour-marked birds to investigate how conspecific density and habitat quality during the tropical non-breeding period interact to affect body condition and apparent annual survival of a long-distance migratory songbird, the American redstart (Setophaga ruticilla). Body condition and survival of birds in high-quality mangrove habitat declined as density increased. By contrast, body condition improved and survival did not vary as density increased in adjacent, lower quality scrub habitat, although mean condition and survival were almost always lower than in mangrove. High rainfall enhanced body condition in scrub but not in mangrove, suggesting factors such as food availability outweighed consequences of crowding in lower quality habitat. Thus, survival of overwintering redstarts in mangrove habitat, disproportionately males, appears to be regulated by a crowding mechanism based on density-dependent resource competition. Survival of individuals in scrub, mostly females, appears to be limited by density-independent environmental factors but not regulated by crowding. The contrasting effects of density and food limitation on individuals overwintering in adjacent habitats illustrate the complexity of processes operating during the non-breeding period for migratory animals, and emphasize the need for long-term studies of animals in multiple habitats and throughout their annual cycles.     

The ratio of acetate‐to‐glucose oxidation in astrocytes from a single 13C NMR spectrum of cerebral cortex

The ¹³C‐labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of ¹³C‐enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α‐ketoglutarate into glutamate in neurons, and incorporation of α‐ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single ¹³C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate‐to‐glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2‐¹³C]acetate and [1,6‐¹³C]glucose, and proton decoupled ¹³C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that ¹³C‐labeled acetate and glucose contributed approximately equally to acetyl‐CoA (0.96) in astrocytes. As this method relies on a single ¹³C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions.