Selective backbone labeling of proteins using {1,2-<Superscript>13</Superscript>C<Subscript>2</Subscript>}-pyruvate as carbon source

A simple isotope labeling approach for selective ¹³C/¹⁵N backbone labeling of proteins is described. Using {1,2-¹³C₂}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of ¹³C^α-¹³CO spin-pairs into the backbones of protein molecules with medium-to-high levels of ¹³C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a 7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein NMR applications are expected to benefit from the {1,2-¹³C₂}-pyruvate based protein production.     

Acetyl-coenzyme A synthase: the case for a Ni<Subscript>p</Subscript><Superscript>0</Superscript>-based mechanism of catalysis

Acetyl-CoA synthase (also known as carbon monoxide dehydrogenase) is a bifunctional Ni-Fe-S-containing enzyme that catalyzes the reversible reduction of CO₂ to CO and the synthesis of acetyl-coenzyme A from CO, CoA, and a methyl group donated by a corrinoid iron-sulfur protein. The active site for the latter reaction, called the A-cluster, consists of an Fe₄S₄ cubane bridged to the proximal Ni site (Ni_p), which is bridged in turn to the so-called distal Ni site. In this review, evidence is presented that Ni_p achieves a zero-valent state at low potentials and during catalysis. Ni_p appears to be the metal to which CO and methyl groups bind and then react to form an acetyl-Ni_p intermediate. Methyl group binding requires reductive activation, where two electrons reduce some site on the A-cluster. The coordination environment of the distal Ni suggests that it could not be stabilized in redox states lower than 2+. The rate at which the [Fe₄S₄]²⁺ cubane is reduced is far slower than that at which reductive activation occurs, suggesting that the cubane is not the site of reduction. An intriguing possibility is that Ni_p²⁺ might be reduced to the zero-valent state. Reinforcing this idea are Ni-organometallic complexes in which the Ni exhibits analogous reactivity properties when reduced to the zero-valent state. A zero-valent Ni stabilized exclusively with biological ligands would be remarkable and unprecedented in biology.Electronic Supplementary Material Supplementary Material is available in the online version of this article at     

Theoretical study of the novel sandwich compound [Au<Subscript>3</Subscript>Cl<Subscript>3</Subscript>Tr<Subscript>2</Subscript>]<Superscript>2+</Superscript>

A theoretical study of a sandwich compound with a metal monolayer sheet between two aromatic ligands is presented. A full geometry optimization of the [Au₃Cl₃Tr₂]²⁺ (1) compound, which is a triangular gold(I) monolayer sheet capped by chlorines and bounded to two cycloheptatrienyl (Tr) ligands was carried out using perturbation theory at the MP2 computational level and DFT. Compound (1) is in agreement with the 18–electron rule, the bonding nature in the complex may be interpreted from the donation interaction coming from the Tr rings to the Au array, and from the back-donation from the latter to the former. NICS calculations show a strong aromatic character in the gold monolayer sheet and Tr ligands; calculations done with HOMA, also report the same aromatic behavior on the cycloheptatrienyl fragments giving us an insight on the stability of (1). The Au –Au bond lengths indicate that an intramolecular aurophilic interaction among the Au(I) cations plays an important role in the bonding of the central metal sheet. Figure (a) Ground state geometry of complex 1; (b) Top view of compound 1 and Wiberg bond orders computed with the MP2/B1 computational method; (c) Lateral view of compound 1 and NICS values calculated with the MP2/B1 method; the values in parenthesis were obtained at the VWN/TZP level     

Salt-induced NO<Subscript>3</Subscript><Superscript>-</Superscript> uptake inhibition in cowpea roots is dependent on the ionic composition of the salt and its osmotic effect

Salinity remarkably inhibits NO₃ ^- uptake but the mechanisms are not well understood. This study was addressed to elucidate the role of ionic and osmotic components of salinity on NO₃ ^- influx and efflux employing classic kinetics involving a low affinity transport system (LATS) and a high affinity transport system (HATS). In the presence of KCl, NaCl, and Na₂SO₄ at 100 mM concentrations, in both LATS and HATS, Michaelis constant (K_m) was similar for the three salts and maximum rate (V_max) decreased as follows: KCl > NaCl > Na₂SO₄, compared to control indicating a non-competitive interaction with NO₃ ^-. Unexpectedly, iso-osmotic solutions (osmotic potential Ψ_π = -0.450) of polyethylene glycol (PEG, 17.84 %, v/v) and mannitol (100 mM) remarkably increased K_m in both the LATS and the HATS, but V_max did not change indicating a competitive inhibition. Under the PEG and mannitol treatments, K_m and V_max were higher than under the salt treatments. The salts increased slightly NO₃ ^- efflux in the following order KCl > NaCl > Na₂SO₄. In contrast, mannitol strongly stimulated and the PEG inhibited NO₃ ^- efflux. The obtained data reveal that salinity effects were not dependent on the anion type (Cl^- versus SO₄ ^2-) indicating a non-competitive inhibition mechanism between Cl^- and NO₃ ^-. In contrast, the cation types (K⁺ versus Na⁺) had a pronounced effect. The osmotic component is important to net NO₃ ^- uptake affecting remarkably the influx in both LATS and HATS components of cowpea roots.     

ADP-Inhibition of H<Superscript>+</Superscript>-F<Subscript>O</Subscript>F<Subscript>1</Subscript>-ATP Synthase

H⁺-F_OF₁-ATP synthase (F-ATPase, F-type ATPase, F_OF₁ complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.     

Catalytic mechanisms of Au<Subscript>11</Subscript> and Au<Subscript>11-n</Subscript>Pt<Subscript>n</Subscript> (<Emphasis Type="Italic">n</Emphasis>=1–2) clusters: a DFT investigation on the oxidation of CO by O<Subscript>2</Subscript>

The oxidation of CO catalyzed by clusters of Au₁₁, Au₁₀Pt and Au₉Pt₂ was investigated using the M06 functional suite of the density functional theory. Au and Pt atoms were described with the double-ζ valence basis set Los Alamos National Laboratory 2-double-z (LanL2DZ), whereas the standard 6-311++G(d,p) basis set was employed for the C and O atoms. Our theoretical model showed that (1) after coordination to Au and Au-Pt cluster, O₂ and CO are apparently activated, and Mulliken charges show that the gold atoms in the active sites of Au11 are negatively charged; (2) Au-Pt clusters with 11 atoms can effectively catalyze the oxidation of CO by O₂; (3) Au₁₁ exhibits good catalytic performance for the oxidation of CO; (4) oxidation of CO occurs preferably on the Au–Pt active sites in Pt-doped clusters, and the single-center mechanisms are more favorable energetically than the two-center mechanisms; (5) after adsorption, an O₂ molecule oxidates two CO molecules via stepwise mechanisms; and (6) the catalytic processes are highly exothermic.     

Comparison of Glutamate Turnover in Nerve Terminals and Brain Tissue During [1,6-<Superscript>13</Superscript>C<Subscript>2</Subscript>]Glucose Metabolism in Anesthetized Rats

The ¹³C turnover of neurotransmitter amino acids (glutamate, GABA and aspartate) were determined from extracts of forebrain nerve terminals and brain homogenate, and fronto-parietal cortex from anesthetized rats undergoing timed infusions of [1,6-¹³C₂]glucose or [2-¹³C]acetate. Nerve terminal ¹³C fractional labeling of glutamate and aspartate was lower than those in whole cortical tissue at all times measured (up to 120 min), suggesting either the presence of a constant dilution flux from an unlabeled substrate or an unlabeled (effectively non-communicating on the measurement timescale) glutamate pool in the nerve terminals. Half times of ¹³C labeling from [1,6-¹³C₂]glucose, as estimated by least squares exponential fitting to the time course data, were longer for nerve terminals (Glu_C4, 21.8 min; GABA_C2 21.0 min) compared to cortical tissue (Glu_C4, 12.4 min; GABA_C2, 14.5 min), except for Asp_C3, which was similar (26.5 vs. 27.0 min). The slower turnover of glutamate in the nerve terminals (but not GABA) compared to the cortex may reflect selective effects of anesthesia on activity-dependent glucose use, which might be more pronounced in the terminals. The ¹³C labeling ratio for glutamate-C4 from [2-¹³C]acetate over that of ¹³C-glucose was twice as large in nerve terminals compared to cortex, suggesting that astroglial glutamine under the ¹³C glucose infusion was the likely source of much of the nerve terminal dilution. The net replenishment of most of the nerve terminal amino acid pools occurs directly via trafficking of astroglial glutamine.     

[HCO<Subscript>3</Subscript><Superscript>-</Superscript>]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

Background  

Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO₃ ^- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl^- permeability. Here, we examined whether HCO₃ ^- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO₃ ^- and the stimulation of CFTR in BCECs.     

Theoretical assessment of oscillations in the concentrations of P<Stack><Subscript>700</Subscript><Superscript>+</Superscript></Stack> and intermediates of the Calvin-Benson cycle

A theoretically obtained oscillatory mode of photosynthesis caused by long-wave illumination is discussed. Periodical variations in the operation of photosystem (PS) I, the electron transport chain, and the Calvin-Benson cycle take place in this mode. The changes in PS II operation are not repetitive. Damped oscillations of P₇₀₀⁺ concentration and CO₂ assimilation rate are observed in the theoretical curves. The results of modeling are quite consistent with the P₇₀₀⁺ kinetics obtained earlier by measuring the induction of the PS I EPR signal in leaves at different temperatures.     

Arbuscular mycorrhiza maintains nodule function during external NH<Stack><Subscript>4</Subscript><Superscript>+</Superscript></Stack> supply in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> (L.)

The synergistic benefits of the dual inoculation of legumes with nodule bacteria and arbuscular mycorrhizae (AM) are well established, but the effect of an external NH₄⁺ supply on this tripartite relationship is less clear. This effect of NH₄⁺ supply was investigated with regards to the growth and function of the legume host and both symbionts. Nodulated Phaseolus vulgaris seedlings with and without AM, were grown in a sand medium with either 0 N, 1 mM or 3 mM NH₄⁺. Plants were harvested at 30 days after emergence and measurements were taken for biomass, N₂ fixation, photosynthesis, asparagine concentration, construction costs and N nutrition. The addition of NH₄⁺ led to a decline in the percentage AM colonization and nodule dry weights, although AM colonization was affected to a lesser extent. NH₄⁺ supply also resulted in a decrease in the reliance on biological nitrogen fixation (BNF); however, the AM roots maintained higher levels of NH₄⁺ uptake than their non-AM counterparts. Furthermore, the non-AM plants had a higher production of asparagine than the AM plants. The inhibitory effects of NH₄⁺ on nodule function can be reduced by the presence of AM at moderate levels of NH₄⁺ (1 mM), via improving nodule growth or relieving the asparagine-induced inhibition of BNF.     

Investigation of S<Subscript>N</Subscript>2 [<Superscript>11</Superscript>C]cyanation for base-sensitive substrates: an improved radiosynthesis of <Emphasis Type="SmallCaps">l</Emphasis>-[5-<Superscript>11</Superscript>C]-glutamine

Carbon-11 (β⁺ emitter, t _1/2 = 20.4 min) radiolabeled l-glutamine is a potentially useful molecular imaging agent that can be utilized with positron emission tomography for both human oncological diagnosis and plant imaging research. Based upon a previously reported [¹¹C]cyanide end-capping labeling method, a systematic investigation of nucleophilic cyanation reactions and acidic hydrolysis reaction parameters, including base, metal ion source, phase transfer catalyst, solvent, reaction temperature and reaction time, was conducted. The result was a milder, more reliable, two-step method which provides l-[5-¹¹C]-glutamine with a radiochemical yield of 63.8 ± 8.7 % (range from 51 to 74 %, n = 10) with >90 % radiochemical purity and >90 % enantiomeric purity. The total synthesis time was 40–50 min from the end of bombardment. In addition, an Fmoc derivatization method was developed to measure the specific activity of this radiotracer.     

Resolution-optimized NMR measurement of <Superscript>1</Superscript>D<Subscript>CH</Subscript>, <Superscript>1</Superscript>D<Subscript>CC</Subscript> and <Superscript>2</Superscript>D<Subscript>CH</Subscript> residual dipolar couplings in nucleic acid bases

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond ²D_CH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in ¹³C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear ¹H-¹H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven ¹H-¹³C and ¹³C-¹³C couplings are measured for pyrimidines (U and C), including ¹D_C5H5, ¹D_C6H6, ²D_C5H6, ²D_C6H5, ¹D_C5C4, ¹D_C5C6, and ²D_C4H5. For adenine, four base couplings (¹D_C2H2, ¹D_C8H8, ¹D_C4C5, and ¹D_C5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (¹D_C8H8, ¹D_C4C5, and ¹D_C5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide.     

Structure determination of proteins in <Superscript>2</Superscript>H<Subscript>2</Subscript>O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment

We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone ¹Hα and ¹³Cα to those of ¹³C′ in the preceding residue. By applying ²H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone ¹³C′ and amide ¹⁵N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for ¹³C/¹⁵N-labeled GB1 dissolved in ²H₂O. The quality of the GB1 structure determined in ²H₂O was similar to that determined in H₂O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in ²H₂O or H₂O at high pH values.     

The expansion of CD4<Superscript>+</Superscript>CD28<Superscript>-</Superscript> T cells in patients with rheumatoid arthritis

Clonal expansion of CD4⁺CD28^- T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4⁺ clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4⁺CD28^- T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4⁺CD28^- T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4⁺CD28^- T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4⁺CD28^- T cells may be a marker correlating with extra-articular manifestations and joint involvement.     

A systematic search for the structures,stabilities, and electronic properties of bimetallic Ca<Subscript>2</Subscript>-doped gold clusters: comparison with pure gold clusters

The local meta-GGA exchange correlation density functional (TPSS) with a relativistic effective core potential was employed to systematically investigate the geometric structures, stabilities, and electronic properties of bimetallic Ca₂Au _n (n = 1–9) and pure gold Au _n (n ≤ 11) clusters. The optimized geometries show that the most stable isomers for Ca₂Au _n clusters have 3D structure when n > 2, and that one Au atom capping the Ca₂Au _n−1 structure for different-sized Ca₂Au _n (n = 1–9) clusters is the dominant growth pattern. The average atomic binding energies and second-order difference in energies show that the Ca₂Au₄ isomer is the most stable among the Ca₂Au _n clusters. The same pronounced even–odd alternations are found in the HOMO–LUMO gaps, VIPs, and hardnesses. The polarizabilities of the Ca₂Au _n clusters show an obvious local minimum at n = 4. Moreover, the inverse corrections to the polarizabilities versus the ionization potential and hardness were found for the gold clusters.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

Pathway-specific metabolome analysis with <Superscript>18</Superscript>O<Subscript>2</Subscript>-labeled <Emphasis Type="Italic">Medicago truncatula</Emphasis> via a mass spectrometry-based approach

Introduction

Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.

Objectives

Pathway-specific labeling using isotopic molecular oxygen (¹⁸O₂) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of ¹⁸O₂-metabolome analysis.

Methods

¹⁸O₂ labeled whole Medicago truncatula seedlings were prepared using ¹⁸O₂-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).

Results

A total of 511 peaks labeled by ¹⁸O₂ from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by ¹⁸O₂. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSⁿ analysis, we estimated that ¹⁸O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an ¹⁸O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an ¹⁸O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.

Conclusion

Our finding indicated that ¹⁸O₂ labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.

     

DNA Detection Based on Localized Surface Plasmon Resonance Spectroscopy of Ag@Au Biocomposite Nanoparticles

Noble metal nanoparticles (NPs) have attracted much attention due to their unique physical and chemical properties such as tunable surface plasmonics, high-efficiency electrochemical sensing, and enhanced fluorescence. We produced two biosensor chips consisting of Ag@Au bimetallic nanoparticles (BNPs) on a carbon thin film by simple RF-sputtering and RF-plasma-enhanced chemical vapor co-deposition. We deposited Au NPs with average size of 4 nm (Au₁ NPs) or 11 nm (Au₂ NPs) on a sensor chip consisting of Ag NPs with mean size of 15 nm, and we investigated the effect of shell size (Au NPs) on the chemical activities of the resulting Ag@Au₁ BNPs and Ag@Au₂ BNPs. We estimated the average size and morphology of Ag@Au BNPs by scanning electron microscopy (SEM) and atomic force microscopy (AFM) images. X-ray diffraction (XRD) patterns revealed that Ag NPs and Au NPs had face-centered cubic (FCC) structure. We studied aging of the biosensor chips consisting of Ag@Au BNPs by localized surface plasmon resonance (LSPR) spectroscopy for up to 3 months. UV–visible aging of the prepared samples indicated that Ag@Au₁ BNPs, which corresponded to Ag NPs covered with smaller Au NPs, were more chemically active than Ag@Au₂ BNPs. Furthermore, we evaluated changes in the LSPR absorption peaks of Ag@Au₁ BNPs and bare Ag NPs in the presence of a DNA primer decamer at fM concentrations, to find that Ag@Au₁ BNPs were more sensitive biosensor chips within a short response time as compared to bare Ag NPs.

     

H<Subscript>2</Subscript> enrichment from synthesis gas by <Emphasis Type="Italic">Desulfotomaculum</Emphasis><Emphasis Type="Italic">carboxydivorans</Emphasis> for potential applications in synthesis gas purification and biodesulfurization

Desulfotomaculum carboxydivorans, recently isolated from a full-scale anaerobic wastewater treatment facility, is a sulfate reducer capable of hydrogenogenic growth on carbon monoxide (CO). In the presence of sulfate, the hydrogen formed is used for sulfate reduction. The organism grows rapidly at 200 kPa CO, pH 7.0, and 55°C, with a generation time of 100 min, producing nearly equimolar amounts of H₂ and CO₂ from CO and H₂O. The high specific CO conversion rates, exceeding 0.8 mol CO (g protein)⁻¹ h⁻¹, makes this bacterium an interesting candidate for a biological alternative of the currently employed chemical catalytic water–gas shift reaction to purify synthesis gas (contains mainly H₂, CO, and CO₂). Furthermore, as D. carboxydivorans is capable of hydrogenotrophic sulfate reduction at partial CO pressures exceeding 100 kPa, it is also a good candidate for biodesulfurization processes using synthesis gas as electron donor at elevated temperatures, e.g., in biological flue gas desulfurization. Although high maximal specific sulfate reduction rates (32 mmol (g protein)⁻¹ h⁻¹) can be obtained, its sulfide tolerance is rather low and pH dependent, i.e., maximally 9 and 5 mM sulfide at pH 7.2 and pH 6.5, respectively.     

Identity of mysterious CD4<Superscript>+</Superscript>CD25<Superscript>-</Superscript>Foxp3<Superscript>+</Superscript>cells in systemic lupus erythematosus

Various abnormalities in CD4⁺CD25⁺ regulatory T cells (Tregs) in systemic lupus erythematosus (SLE) include increased Foxp3⁺ cells that are CD25 negative. Barring methodological technical factors, these cells could be atypical Tregs or activated non-Treg CD4⁺ cells that express Foxp3. Two groups have reached opposite conclusions that could possibly reflect the subjects studied. One group studied untreated new-onset SLE and suggested that these T cells were mostly CD25^-Foxp3⁺ non-Tregs. The other group studied patients with long-standing disease and suggested that these cells are mostly dysfunctional Tregs. A third group reported increased Foxp3⁺CD4⁺CD25^dim rather than CD25^- cells in active SLE and these were also non-Tregs. Thus, it is likely that not all Foxp3⁺ T cells in SLE have protective suppressive activity.