Increased risk of latent tuberculous infection among persons with pre-diabetes and diabetes mellitus.

SETTING: Although diabetes mellitus (DM) is an established risk factor for active tuberculosis (TB) disease, little is known about the association between pre-DM, DM, and latent tuberculous infection (LTBI). OBJECTIVE: To estimate the association between DM and LTBI. DESIGN: We conducted a cross-sectional study among recently arrived refugees seen at a health clinic in Atlanta, GA, USA, between 2013 and 2014. Patients were screened for DM using glycosylated-hemoglobin (HbA1c), and for LTBI using the QuantiFERON(®)-TB (QFT) test. HbA1c and QFT results, demographic information, and medical history were abstracted from patient charts. RESULTS: Among 702 included patients, 681 (97.0%) had HbA1c and QFT results. Overall, 54 (7.8%) patients had DM and 235 (33.8%) had pre-DM. LTBI was prevalent in 31.3% of the refugees. LTBI prevalence was significantly higher (P < 0.01) among patients with DM (43.4%) and pre-DM (39.1%) than in those without DM (25.9%). Refugees with DM (adjusted OR [aOR] 2.3, 95%CI 1.2-4.5) and pre-DM (aOR 1.7, 95%CI 1.1-2.4) were more likely to have LTBI than those without DM. CONCLUSION: Refugees with DM or pre-DM from high TB burden countries were more likely to have LTBI than those without DM. Dysglycemia may impair the immune defenses involved in preventing Mycobacterium tuberculosis infection.

Directed assembly of nanoparticles to isolated diatom valves using the non-wetting characteristics after pyrolysis.

A novel strategy for a directed nanoparticle coupling to isolated Stephanopyxis turris valves is presented. After pyrolysis, the valves exhibit incomplete wetting due to their characteristic T-shaped profiles as a prerequisite for a regioselective coupling reaction. A micromanipulation system allows for precise handling and their immobilization onto an adhesive substrate and manipulation into arrays.

Structure, function and evolution of the Archaeal class I fructose-1,6-bisphosphate aldolase.

FBPA (fructose-1,6-bisphosphate aldolase) catalyses the reversible aldol condensation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate to form fructose 1,6-bisphosphate. Two classes of FBPA, which rely on different reaction mechanisms, have so far been discovered, class I mainly found in Eucarya and class II mainly in Bacteria. Only recently were genes encoding proteins with FBPA activity identified in Archaea. Archaeal FBPAs do not share any significant overall sequence identity with members of the traditional classes of FBPAs, raising the interesting question of whether they have evolved independently by convergent evolution or diverged from a common ancestor. Biochemical characterization of FBPAs of the two hyperthermophilic Archaea Thermoproteus tenax and Pyrococcus furiosus showed that the enzymes use a Schiff-base mechanism and thus belong to the class I aldolases. The crystal structure of the archaeal FBPA from T. tenax revealed that the protein fold, as for the classical FBPA I and II, is that of a parallel (betaalpha)(8) barrel. A substrate-bound crystal structure allowed detailed active-site comparisons which showed the conservation of six important catalytic and substrate-binding residues between the archaeal and the classical FBPA I. This observation provides further evidence that the two sequence families of proteins share a common evolutionary origin. Furthermore, structure and sequence analysis indicate that the class I FBPA shares a common evolutionary origin with several other enzyme superfamilies of the (betaalpha)(8) barrel fold.

Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in Thermoproteus tenax: metabolic parallelism or specific adaptation?

Genome data as well as biochemical studies have indicated that--as a peculiarity within hyperthermophilic Archaea--Thermoproteus tenax uses three different pathways for glucose metabolism, a variant of the reversible EMP (Embden-Meyerhof-Parnas) pathway and two different modifications of the ED (Entner-Doudoroff) pathway, a non-phosphorylative and a semi-phosphorylative version. An overview of the three different pathways is presented and the physiological function of the variants is discussed.

Triosephosphate isomerase of the hyperthermophile Thermoproteus tenax: thermostability is not everything.

The triosephosphate isomerase of the hyperthermophilic crenarchaeum Thermoproteus tenax (TtxTIM) represents a homomeric tetramer. Unlike the triosephosphate isomerases of other hyperthermophiles, however, the association of the TtxTIM tetramers is looser, allowing a reversible dissociation into inactive dimers. The dimer/tetramer equilibrium of TtxTIM is shifted to the tetrameric state through a specific interaction with glycerol-1-phosphate dehydrogenase of T. tenax, suggesting that higher oligomerization of the TtxTIM serves functional rather than stabilizing purposes.

Role of two different glyceraldehyde-3-phosphate dehydrogenases in controlling the reversible Embden-Meyerhof-Parnas pathway in Thermoproteus tenax: regulation on protein and transcript level.

The hyperthermophilic archaeum Thermoproteus tenax uses a variant of the Embden-Meyerhof-Parnas (EMP) pathway as the main route for carbohydrate metabolism. This variant is characterized by a reversible nonallosteric PPi-dependent phosphofructokinase and two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity, phosphate dependence, and allosteric behavior. Although the nonphosphorylating NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN; E.C. 1.2.1.8) fulfills exclusively catabolic purposes, the phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP+-GAPDH; E.C. 1.2.1.13) exhibits anabolic features. The gene encoding the NADP+-GAPDH was cloned, sequenced, and expressed in Escherichia coli. The deduced protein sequence displayed 47%-53% sequence identity to archaeal phosphorylating GAPDHs. The kinetic parameters of the NADP+-GAPDH showed a clear preference for the reductive reaction with a 5-fold-higher specific activity in the reductive reaction as compared to the oxidative reaction and a 20-fold-lower Km for 1,3-bisphosphoglycerate as compared to glyceraldehyde-3-phosphate. Contrary to GAPN, the enzyme is not allosterically regulated. The coding gene overlaps by 1 bp with a preceding open reading frame coding for 3-phosphoglycerate kinase (PGK; E.C. 2.7.2.3). Northern analyses identified mono- and bicistronic messages of both genes in an equimolar ratio. Transcript levels and specific activity of NADP+-GAPDH and PGK were 3- to 4-fold higher under autotrophic conditions as compared to heterotrophic conditions, whereas transcript abundance and specific activity of GAPN remained constant in autotrophically and heterotrophically grown cells. The different regulation of the two counteracting glyceraldehyde-3-phosphate dehydrogenases is discussed with respect to the flux control of the T. tenax-specific EMP variant.

Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase.

Comparative structural studies on proteins derived from organisms with growth optima ranging from 15 to 100 degrees C are beginning to shed light on the mechanisms of protein thermoadaptation. One means of sustaining hyperthermostability is for proteins to exist in higher oligomeric forms than their mesophilic homologues. Triosephosphate isomerase (TIM) is one of the most studied enzymes, whose fold represents one of nature's most common protein architectures. Most TIMs are dimers of approximately 250 amino acid residues per monomer. Here, we report the 2.7 A resolution crystal structure of the extremely thermostable TIM from Pyrococcus woesei, a hyperthermophilic archaeon growing optimally at 100 degrees C, representing the first archaeal TIM structure. P. woesei TIM exists as a tetramer comprising monomers of only 228 amino acid residues. Structural comparisons with other less thermostable TIMs show that although the central beta-barrel is largely conserved, severe pruning of several helices and truncation of some loops give rise to a much more compact monomer in the small hyperthermophilic TIM. The classical TIM dimer formation is conserved in P. woesei TIM. The extreme thermostability of PwTIM appears to be achieved by the creation of a compact tetramer where two classical TIM dimers interact via an extensive hydrophobic interface. The tetramer is formed through largely hydrophobic interactions between some of the pruned helical regions. The equivalent helical regions in less thermostable dimeric TIMs represent regions of high average temperature factor. The PwTIM seems to have removed these regions of potential instability in the formation of the tetramer. This study of PwTIM provides further support for the role of higher oligomerisation states in extreme thermal stabilisation.

Production of volatile derivatives of metal(loid)s by microflora involved in anaerobic digestion of sewage sludge.

Gases released from anaerobic wastewater treatment facilities contain considerable amounts of volatile methyl and hydride derivatives of metals and metalloids, such as arsine (AsH(3)), monomethylarsine, dimethylarsine, trimethylarsine, trimethylbismuth (TMBi), elemental mercury (Hg(0)), trimethylstibine, dimethyltellurium, and tetramethyltin. Most of these compounds could be shown to be produced by pure cultures of microorganisms which are representatives of the anaerobic sewage sludge microflora, i.e., methanogenic archaea (Methanobacterium formicicum, Methanosarcina barkeri, Methanobacterium thermoautotrophicum), sulfate-reducing bacteria (Desulfovibrio vulgaris, D. gigas), and a peptolytic bacterium (Clostridium collagenovorans). Additionally, dimethylselenium and dimethyldiselenium could be detected in the headspace of most of the pure cultures. This is the first report of the production of TMBi, stibine, monomethylstibine, and dimethylstibine by a pure culture of M. formicicum.

A mass balance study to evaluate the biotransformation and excretion of [14C]-triamcinolone acetonide following oral administration.

The principle objective of this study was to characterize the absorption, metabolism, and disposition of orally administered [14C]-triamcinolone acetonide. Six healthy male subjects each received a single 100 microCi (approximately 800 micrograms) oral dose of [14C]-triamcinolone acetonide. Plasma, urine, and fecal samples were collected at selected times and analyzed for triamcinolone acetonide and [14C]-derived radioactivity. Plasma protein binding of triamcinolone acetonide was also determined. Metabolite profiling and identification were carried out in plasma and excreta. Principle metabolites were assessed for activity with in vitro anti-inflammatory models. [14C]-triamcinolone acetonide was found to be systemically absorbed following oral administration. The presystemic metabolism and clearance of triamcinolone acetonide were extensive, with only a small fraction of the total plasma radioactivity being made up of triamcinolone acetonide. Little to no parent compound was detected in the plasma 24 hours after administration. Most of the urinary and fecally [14C]-derived radioactivity was also excreted within 24 and 72 hours postdose, respectively. Mean plasma protein binding of triamcinolone acetonide was constant, predictable, and a relatively low 68% over a 24-fold range of plasma concentrations. Three principle metabolites of triamcinolone acetonide were profiled in plasma, urine, and feces. These metabolites were identified as 6 beta-hydroxy triamcinolone, 21-carboxylic acid triamcinolone acetonide, and 6 beta-hydroxy-21-oic triamcinolone acetonide. All three metabolites failed to show any concentration-dependent effects in anti-inflammatory models evaluating IL-5-sustained eosinophil viability and IgE-induced basophil histamine release.

Pyruvate kinase of the hyperthermophilic crenarchaeote Thermoproteus tenax: physiological role and phylogenetic aspects.

Pyruvate kinase (PK; EC 2.7.1.40) of Thermoproteus tenax was purified to homogeneity, and its coding gene was cloned and expressed in Escherichia coli. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg(2+) and Mn(2+). Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K(+) ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the T. tenax-specific variant of the Embden-Meyerhof-Parnas pathway. T. tenax PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gram-positive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in Archaea, show activity without effectors but are commonly regulated by the energy charge of the cell.

Crystallization and preliminary X-ray diffraction analysis of the NAD-dependent non--phosphorylating GAPDH of the hyperthermophilic archaeon Thermoproteus tenax.

Recombinant non-phosphorylating NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic crenarchaeote Thermoproteus tenax has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion technique. Crystals of different habits were obtained from several precipitant solutions (salts and polyethylene glycols). Preliminary X-ray analysis was performed with crystals grown in ammonium formate, which belonged to the primitive hexagonal space group P622, and had unit-cell parameters a = b = 184.8, c = 133.0 A, gamma = 120 degrees. Assuming a molecular weight of 55 kDa, a Matthews parameter of 3.3 A(3) Da(-1) is calculated assuming two molecules per asymmetric unit. The diffraction limit of these crystals is 2.5 A resolution.

Microbial degradation of octamethylcyclotetrasiloxane.

The microbial degradation of low-molecular-weight polydimethylsiloxanes was investigated through laboratory experiments. Octamethylcyclotetrasiloxane was found to be biodegraded under anaerobic conditions in composted sewage sludge, as monitored by the occurrence of the main polydimethylsiloxane degradation product, dimethylsilanediol, compared to that found in experiments with sterilized control samples.

Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus.

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.

NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties.

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.

PPi-dependent phosphofructokinase from Thermoproteus tenax, an archaeal descendant of an ancient line in phosphofructokinase evolution.

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PPi-dependent PFK (PPi-PFK; EC 2.7.1.90), which uses PPi instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PPi-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The approximately 100-kDa PPi-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PPi-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PPi-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PPi as a cosubstrate.

Preliminary crystallographic studies of triosephosphate isomerase (TIM) from the hyperthermophilic Archaeon Pyrococcus woesei.

Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 A and beta = 92.8 degrees, and diffract to at least 2.6 A. The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 A, and diffract to 2.9 A. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centering. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to our understanding of protein thermostability.

Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase.

Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the 13C label in alanine synthesized by cells grown with [1-13C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD+ and NADP+ as cosubstrates, showing a 100-fold higher affinity for NADP+. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP+. A striking feature was the influence of NADP+ and NAD+ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases.