Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol

In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion and are synthesized by a conserved enzymatic pathway that includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine, directly forming N-acetylneuraminic acid (or sialic acid). In this paper we report the development of a coupled assay to monitor NeuB reaction kinetics and an 18O-labeling study that demonstrates the synthase operates via a C-O bond cleavage mechanism. We also report the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (beta/alpha)8 barrel (TIM barrel)-type fold at the N-terminal end and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. We have determined the structures of NeuB in the malate-bound form and with bound PEP and the substrate analog N-acetylmannosaminitol to 1.9 and 2.2 A resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature that leads to distinct modes of substrate binding from other PEP-utilizing enzymes that lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered manganese and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the N-acetylmannosamine carbonyl to the addition of PEP.     

Pharmacokinetics and pharmacodynamics of intravenous artesunate during severe malaria treatment in Ugandan adults

ABSTRACT: BACKGROUND: Severe malaria is a medical emergency with high mortality. Prompt achievement of therapeutic concentrations of highly effective anti-malarial drugs reduces the risk of death. The aim of this study was to assess the pharmacokinetics and pharmacodynamics of intravenous artesunate in Ugandan adults with severe malaria. METHODS: Fourteen adults with severe falciparum malaria requiring parenteral therapy were treated with 2.4 mg/kg intravenous artesunate. Blood samples were collected after the initial dose and plasma concentrations of artesunate and dihydroartemisinin measured by solid-phase extraction and liquid chromatography-tandem mass spectrometry. The study was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (Ref2010-015) and Uganda National Council of Science and Technology (HS605) and registered with ClinicalTrials.gov (NCT01122134). RESULTS: All study participants achieved prompt resolution of symptoms and complete parasite clearance with median (range) parasite clearance time of 17 (8-24) hours. Median (range) maximal artesunate concentration (Cmax) was 3260 (1020-164000) ng/mL, terminal elimination half-life (T1/2) was 0.25 (0.1-1.8) hours and total artesunate exposure (AUC) was 727 (290-111256) ngh/mL. Median (range) dihydroartemisinin Cmax was 3140 (1670-9530) ng/mL, with Tmax of 0.14 (0.6 - 6.07) hours and T1/2 of 1.31 (0.8-2.8) hours. Dihydroartemisinin AUC was 3492 (2183-6338) ngh/mL. None of the participants reported adverse events. CONCLUSIONS: Plasma concentrations of artesunate and dihydroartemisinin were achieved rapidly with rapid and complete symptom resolution and parasite clearance with no adverse events.     

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.     

A Cell Culture Model for Androgen Effects in Motor Neurons

Abstract: Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.     

Fill Factor Losses in Cu2ZnSn(SxSe1−x)4 Solar Cells: Insights from Physical and Electrical Characterization of Devices and Exfoliated Films

Besides the open circuit voltage (V_OC) deficit, fill factor (FF) is the second most significant parameter deficit for earth‐abundant kesterite solar cell technology. Here, various pathways for FF loss are discussed, with focus on the series resistance issue and its various contributing factors. Electrical and physical characterizations of the full range of bandgap (E_g = 1.0–1.5 eV) Cu₂ZnSn(S_xSe_1?x)₄ (CZTSSe) devices, as well as bare and exfoliated films with various S/(S + Se) ratios, are performed. High intensity Suns‐V_OC measurement indicates a nonohmic junction developing in high bandgap CZTSSe. Grazing incidence X‐ray diffraction, Raman mapping, field emission scanning electron microscopy, and X‐ray photoelectron spectroscopy indicate the formation of Sn(S,Se)₂, Mo(S,Se)₂, and Zn(S,Se) at the high bandgap CZTSSe/Mo interface, contributing to the increased series resistance (R_S) and nonohmic back contact characteristics. This study offers some clues as to why the record‐CZTSSe solar cells occur within a bandgap range centered around 1.15 eV and offers some direction for further optimization.     

Anti-kinetochore antibodies: use as probes for inactive centromeres.

Application of a modified immunofluorescence technique using an anti-kinetochore serum enables cytogeneticists to obtain quality metaphase spreads and to localize kinetochores. In a patient with a 45, XX, -9, -11, tdic (9p;11p) constitution, we found that the dicentric marker chromosome has an intensely fluorescent kinetochore (no. 11), the functional centromere, and a less intensely fluorescent kinetochore (no. 9), the inactive centromere. The data suggest that in the process of tandem fusion (telomere-telomere between 11p and 9p), the centromere of chromosome 9 was not deleted, but, rather, inactivated.     

Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle

Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.