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.

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.

Primary structure of glyceraldehyde-3-phosphate dehydrogenase deduced from the nucleotide sequence of the thermophilic archaebacterium Methanothermus fervidus.

The gene for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the thermophilic methanogenic archaebacterium Methanothermus fervidus (growth optimum at 84 degrees C) was cloned in Escherichia coli and the nucleotide sequence was determined. A striking preference for adenine and thymidine bases was found in the gene, which is in agreement with the low G + C content of the M. fervidus DNA. The deduced amino acid sequence indicates an Mr of 37,500 for the protein subunit. Alignment with the amino acid sequences of GAPDHs from other organisms shows that the archaebacterial GAPDH is homologous to the respective eubacterial and eukaryotic enzymes, but the similarity between the archaebacterial enzyme and the eubacterial or eukaryotic GAPDHs is much less than that between the latter two.

The 3-phosphoglycerate kinase of the hyperthermophilic archaeum Pyrococcus woesei produced in Escherichia coli: loss of heat resistance due to internal translation initiation and its restoration by site-directed mutagenesis.

Heterologous expression of the gene coding for 3-phosphoglycerate kinase (PGK) of the hyperthermophilic archaeum, Pyrococcus woesei (Pw), in Escherichia coli (Ec) yielded only low recovery of recombinant PGK (re-PGK) in heat-precipitated crude extracts. Moreover, we noticed contamination with a 28-kDa protein, from which PGK could hardly be separated, even under stringent conditions after tagging the re-PGK with a His6-tag. The preparations contaminated with the 28-kDa protein showed an unexpectedly low thermal stability. Under the same conditions (85 degrees C, 30 min), however, the enzyme from the original organism was completely resistant to heat inactivation. As shown by size-exclusion chromatography, re-PGK forms tight associations with the 28-kDa protein, which was found to represent a C-terminal fragment of PGK and to arise as a product of internal translation initiation within the pgk gene. Mutations changing the internal ribosome-binding site effectively suppressed the production of the 28-kDa protein and restored the thermal stability of the Pw re-PGK.

Cloning and sequencing the gene encoding 3-phosphoglycerate kinase from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus.

The nucleotide sequences of the gene (pgk) encoding 3-phosphoglycerate kinase (PGK) from the mesophilic archaebacterium, Methanobacterium bryantii, and from the closely related thermophile, Methanothermus fervidus, were determined. The deduced amino acid (aa) sequences show 61% identity with each other and 32-36% identity with the enzyme homologues from eubacteria and eukaryotes. As found for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-malate dehydrogenase, the relatedness between the archaebacterial aa sequences on the one hand and the eubacterial or eukaryotic sequences on the other is lower than that between the latter ones. Comparison of the aa sequence of PGK from mesophilic and thermophilic archaebacteria indicates an increase of the overall hydrophobicity and a decrease of the chain flexibility in the thermophilic enzyme, as already deduced from respective comparisons between GAPDH aa sequences of the same organisms. In addition, glycine residues are strikingly discriminated in the thermophilic PGK, which was also observed for GAPDH. Contrary to GAPDH, however, Lys and Arg residues are preferred in the thermophilic PGK. Lys to Arg substitutions are the most frequent cold-to-hot changes in PGK, whereas in GAPDH from the same organisms these changes do not occur.

Tetrameric triosephosphate isomerase from hyperthermophilic Archaea.

Triosephosphate isomerase (TIM) of the hyperthermophilic Archaea Pyrococcus woesei and Methanothermus fervidus have been purified to homogeneity. The enzymes from the two hyperthermophiles represent homo-tetramers of 100 kDa, contrary to all known bacterial and eukaryotic TIMs, which are dimers of 48-60 kDa. Molecular size determination of the TIM from the mesophilic methanogen Methanobacterium bryantii yielded the usual molecular mass of only 57 kDa, indicating that the tetrameric aggregation state does not represent an archaeal feature but rather correlates with thermoadaptation. A similar preference for higher protein aggregates in hyperthermophilic Archaea has previously been demonstrated for 3-phosphoglycerate kinases. The gene of the P. woesei TIM was cloned and sequenced. The archaeal TIM proved to be homologous to its bacterial and eukaryotic pendants. Most strikingly, the deduced protein sequence comprises only 224 residues and thus represents the shortest TIM sequence known as yet. Taking the three-dimensional structure of the eucaryal TIM as a basis, from the shortenings of the chain considerable rearrangements at the bottom of the alpha/beta barrel and at its functionally inactive flank are expected, which are interpreted in terms of the formation of new subunit contacts.

Expression of the glyceraldehyde-3-phosphate dehydrogenase gene from the extremely thermophilic archaebacterium Methanothermus fervidus in E. coli. Enzyme purification, crystallization, and preliminary crystal data.

The gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extremely thermophilic archaebacterium Methanothermus fervidus (growth optimum 82 degrees C) was cloned in vector pJF118EH and expressed in E. coli cells. As shown by molecular mass determination, protein sequencing, heat stability, and substrate saturation kinetics, the enzyme synthesized in E. coli is identical to the original enzyme from M. fervidus. The high thermostability of the E. coli-produced M. fervidus GAPDH allows rapid purification to homogeneity. From this enzyme protein crystals were grown which proved to be suitable for X-ray analysis. The crystals are of tetragonal space group P4(1)22 and contain a dimer per asymmetric unit.

Di-myo-inositol-1,1'-phosphate: a new inositol phosphate isolated from Pyrococcus woesei.

A new inositol derivative could be isolated from the Archaeum Pyrococcus woesei and identified as di-myo-inositol-1,1'-phosphate by 1H, 31P NMR spectroscopy, mass spectrometry and thin layer chromatography. In P. woesei, this inositol phosphate represents the dominant counterion of K+ which ranges from 500 to 600 mM. The role of the potassium salt of di-myo-inositol-1,1'-phosphate as thermostabilizer is discussed.

Biosynthesis of cyclic 2,3-diphosphoglycerate. Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus.

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.

Engineering thermostability in archaebacterial glyceraldehyde-3-phosphate dehydrogenase. Hints for the important role of interdomain contacts in stabilizing protein conformation.

Construction of hybrid enzymes between the glyceraldehyde-3-phosphate dehydrogenases from the mesophilic Methanobacterium bryantii and the thermophilic Methanothermus fervidus by recombinant DNA techniques revealed that a short C-terminal fragment of the Mt. fervidus enzyme contributes largely to its thermostability. This C-terminal region appears to be homologous to the alpha 3-helix of eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenases which is involved in the contacts between the two domains of the enzyme subunit. Site-directed mutagenesis experiments indicate that hydrophobic interactions play an important role in these contacts.

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.

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.

Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus.

The D-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the D-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD+ and NADP+ and is not inhibited by pentalenolactone. The enzyme is intrinsically stable up to 75 degrees C. It is stabilized by the coenzyme NADP+ and at high ionic strength up to about 90 degrees C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52 degrees C.

Cloning, sequencing and expression of the gene encoding 2-phosphoglycerate kinase from Methanothermus fervidus.

The gene encoding 2-phosphoglycerate kinase (2PGK), which catalyses the first step in the biosynthesis of cyclic 2,3-diphosphoglycerate in methanogens, was cloned and sequenced from the hyperthermophilic Methanothermus fervidus. The 2pgk gene codes for 304 amino acids, corresponding to a relative molecular mass of 35040. The 2pgk mRNA was estimated to be 1600 nucleotides in size. Putative transcription signals and the ribosome-binding site of 2pgk are discussed. Production of 2PGK from M. fervidus in Es-cherichia coli reveals the same apparent molecular weights for the native enzyme and its denatured subunit as those shown by the 2PGK purified from M. fervidus. Also the kinetic parameters of 2PKG produced in E. coli correspond well with those from the enzyme isolated from the natural host M. fervidus.

Factors affecting the quaternary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei and Lactobacillus curvatus as investigated by hybridization and ultracentrifugation.

The allosteric L-lactate dehydrogenases of Lactobacillus curvatus and Lactobacillus casei exist in the tetrameric from (molecular weight about 145 000) at pH 5.0--5.5 even in the absence of the effectors Mn2+ and Fru(1,6)P2 (fructose 1,6-bisphosphate), but undergo reversible dissociation to monomers (molecular weight about 35 000) at higher pH values or in the presence of urea. In the range between pH 5.5 (tetrameric state) and pH 7.4 (monomeric state) the L. curvatus L-lactate dehydrogenase exists in a dissociation-association equilibrium comprising tetramers, dimers and monomers as indicated by the Sc20,w values and the results of hybridization experiments. The simultaneous addition of both effectors [Mn2+ and Fru(1,6)P2] at pH 7.4, however, resulted in the stabilization of the tetrameric form. The addition of Fru(1,6)P2 alone at pH 7.4 had almost no influence on the quarternary structure, whereas the addition of Mn2+, as well as that of NADH, largely prevented dissociation. The L-lactate dehydrogenase of L. casei showed similar properties, although the enzyme dissociates only at about pH greater than or equal to 7.8. As in the case of the L. curvatus enzyme, Fru(1,6)P2 has no influence on the pH-dependent dissociation of the L. casei enzyme, whereas Mn2+ stabilizes the tetrameric structure. Reconstitution of a mixture of the two dissociated enzymes results in the formation of all statistically possible, enzymatically active hybrids. No hybridization between the allosteric enzymes from L. casei and L. curvatus and the non-allosteric ones of Lactobacillus plantarum and Lactobacillus acidophilus was observed.