Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS–PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 Å. The crystal belonged to space group C222₁, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 Å, and α = β = γ = 90°.     

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis e virus

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T = 3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~ 350 Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~ 440 Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. &; Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA 108, 12681–12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.     

Expression,purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69 mg per 20 L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3 mg per 10 L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K_m and k_cat) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4 Å. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.     

Soil water utilization characteristics of Haloxylon ammodendron plantation with different age during summer

Haloxylon ammodendron is one of the main shrubs that were used in desertification control project in China. Large area (2700 km²) of H. ammodendron plantation, especially more than 10 years plantation, has degraded outside Minqin oasis, northwest China. It is hard for H. ammodendron plantation to utilize ground water deeper than 20 m and to use precipitation with only mean annual 116.2 mm and uneven distribution in growing season. Thus, soil water might be the main water source of H. ammodendron plantation. Moreover, following H. ammodendron grows up, more soil water will be needed. In this study, it is hypothesized that H. ammodendron plantation would utilize deeper soil water as its age increased. Water use characteristics of different ages of H. ammodendron plantation (2 years, 5 years, 10 years, 20 years and 30 years) were examined by stable oxygen isotope technology outside Minqin oasis in July 2009. The δ¹⁸O values of water in H. ammodendron xylem, six different depths of soil (20 cm, 30 cm, 50 cm, 100 cm, 150 cm and 200 cm), rain and ground water (replaced by well water) were compared to determine major water source of H. ammodendron plantation. Meanwhile, the density, height, length and width of individuals were measured, and soil water contents were examined at the six depths in these plantation. The results showed that in 5 years H. ammodendron plantation, soil water content in 50 cm was significantly lower than moving sand dune and other ages, and soil water content in 100–200 cm was lower than moving sand dune and 2 years H. ammodendron plantation. The main depth that H. ammodendron used soil water increased as the age increased; 2 years H. ammodendron mainly used 50–200 cm soil water; 5 years H. ammodendron used 100–200 cm soil water and started to use ground water; 10 years H. ammodendron used 150–200 cm soil water and ground water, plant density of H. ammodendron declined and soil water content recovered gradually; 20 years and 30 years H. ammodendron mainly used ground water. Therefore, excessive consumption to soil water of high plant density might be one of the reasons for the degradation of H. ammodendron plantation. The critical age of H. ammodendron plantation is 5 years outside Minqin oasis because it consumed excessive soil water. If the density of 5 years H. ammodendron plantation was not decreased by selective cutting at present, it would degrade as the stand age increased later. It is suggested that initial density of new H. ammodendron plantation outside Minqin oasis must be reduced to slow down its consumption of soil water in future, and then the stability of H. ammodendron plantation could be sustained for a longer time.     

Structure of a dimeric fungal α-type carbonic anhydrase

The crystal structure of Aspergillus oryzae carbonic anhydrase (AoCA) was determined at 2.7 Å resolution and it revealed a dimer, which only has precedents in the α class in two membrane and cancer-associated enzymes. α carbonic anhydrases are underrepresented in fungi compared to the β class, this being the first structural representative. The overall fold and zinc binding site resemble other well studied carbonic anhydrases. A major difference is that the histidine, thought to be the major proton shuttle residue in most mammalian enzymes, is replaced by a phenylalanine in AoCA. This finding poses intriguing questions as to the biological functions of fungal α carbonic anhydrases, which are promising candidates for biotechnological applications.Structured summaryAoCA binds to AoCA by molecular sieving (View interaction)AoCA binds to AoCA X-ray crystallography (View interaction)     

Prevotella bivia as a source of lipopolysaccharide in the vagina

ObjectivesTo compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA).MethodsVaginal washes obtained from patients with (n = 43) and without (n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts.ResultsThe median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log₁₀ CFU/ml, respectively, P < 0.001).Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS).The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV.ConclusionP. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.     

Application of carbon fiber composite minielectrodes for measurement of kinetic constants of nitric oxide decay in solution

Carbon fiber microelectrodes and carbon fiber composite minielectrodes (CFM/CFCM) have been generally used for measurements of nitric oxide (NO) concentration in chemical and biological systems. The response time of a CFM/CFCM is usually from milliseconds to seconds depending on the electrode size, the thickness of coating layers on the electrode, and NO diffusion coefficients of the coating layers. As a result, the time course of recoded current changes (I–t curves) by the CFM/CFCM may be different from the actual time course of NO concentration changes (c–t curves) if the half-life of NO decay is close to or shorter than the response time of the electrode used. This adds complexity to the process for determining rate constants of NO decay kinetics from the recorded current curves (I–t curves). By computer simulations based on a mathematical model, an approximation method was developed for determining rate constants of NO decay from the recorded current curves. This method was first tested and valuated using a commercial CFCM in several simple reaction systems with known rate constants. The response time of the CFCM was measured as 4.7 ± 0.7 s (n = 5). The determined rate constants of NO volatilization and NO autoxidation in our measurement system at 37 °C are (1.9 ± 0.1) × 10^?3 s^?1 (n = 4) and (2.0 ± 0.3) × 10³ M^?1 s^?1 (n = 7), which are close to the reported rate constants. The method was then applied to determine the rate of NO decay in blood samples from control and smoking exposed mice. It was observed that the NO decay rate in the smoking group is >20% higher than that in control group, and the increased NO decay rate in the smoking group was reversed by 10 μM diphenyleneiodonium chloride (DPI), an inhibitor of flavin enzymes such as leukocyte NADPH oxidase.     

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

N-1 and C-3 substituted indole Schiff bases as selective COX-2 inhibitors: synthesis and biological evaluation

A group of N-1 and C-3 disubstituted-indole Schiff bases bearing an indole N-1 (R′ = H, CH₂Ph, COPh) substituent in conjunction with a C-3 –CHN–C₆H₄–4-X (X = F, Me, CF₃, Cl) substituent were synthesized and evaluated as inhibitors of cyclooxygenase (COX) isozymes (COX-1/COX-2). Within this group of Schiff bases, compounds 15 (R¹ = CH₂Ph, X = F), 17 (R¹ = CH₂Ph, X = CF₃), 18 (R¹ = COPh, X = F) and 20 (R¹ = COPh, X = CF₃) were identified as effective and selective COX-2 inhibitors (COX-2 IC₅₀’s = 0.32–0.84 μM range; COX-2 selectivity index (SI) = 113 to >312 range). 1-Benzoyl-3-[(4-trifluoromethylphenylimino)methyl]indole (20) emerged as the most potent (COX-1 IC₅₀ >100 μM; COX-2 IC₅₀ = 0.32 μM) and selective (SI >312) COX-2 inhibitor. Furthermore, compound 20 is a selective COX-2 inhibitor in contrast to the reference drug indomethacin that is a potent and selective COX-1 inhibitor (COX-1 IC₅₀ = 0.13 μM; COX-2 IC₅₀ = 6.9 μM, COX-2 SI = 0.02). Molecular modeling studies employing compound 20 showed that the phenyl CF₃ substituent attached to the CN spacer is positioned near the secondary pocket of the COX-2 active site, the CN nitrogen atom is hydrogen bonded (N?NH = 2.85 Å) to the H90 residue, and the indole N-1 benzoyl is positioned in a hydrophobic pocket of the COX-2 active site near W387.     

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters

AimsLate phase ischemic preconditioning (LPC) protects the heart against ischemia–reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism.Main methodsC57BL/6 mice were subjected to 30 min coronary ligation followed by 60 min or 24 h reperfusion with or without LPC (3 cycles of 5 min I/5 min R): Sham, LPC, I/R, and LPC + I/R group. Myocardial tissue Po₂ and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy.Key findingsUpon reperfusion, tissue Po₂ rose significantly above the pre-ischemic level in the I/R mice (23.1 ± 2.2 vs. 12.6 ± 1.3 mm Hg, p < 0.01). This hyperoxygenation was attenuated by LPC in the LPC + I/R mice (11.9 ± 2.0 mm Hg, p < 0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC + I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC + I/R mice compared to that in the Sham control (1.24 ± 0.01 and 1.23 ± 0.01, p < 0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268 ± 0.0016/min) and was corrected by LPC in the LPC + I/R mice (0.0379 ± 0.0023/min). Finally, LPC reduced the infarct size in the LPC + I/R mice (10.5 ± 0.4% vs. 33.3 ± 0.6%, p < 0.05).SignificanceThus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.     

An extract based on the medicinal mushroom Agaricus blazei Murill stimulates monocyte-derived dendritic cells to cytokine and chemokine production in vitro

Background: There is a growing body of evidence that physical training exerts its potential benefits on the individual health status by modulating the immune system and the whole body metabolism. A better knowledge of the physiologic immune response to exercise may help to understand the benefits of physical exercise in healthy individuals and elite athletes. Aims: This study aims to analyse cardiotrophin-1 (CT-1) and Tumor Necrosis Factor-α (TNF-α) plasma levels at rest and during exercise in elite athletes and healthy controls. Methods: We studied 20 triathletes (TA) and 20 matched controls (CG). Chambers dimensions, left ventricular mass and left ventricular mass index were analysed by echocardiography. VO2 peak and VE/VCO2 were calculated by metabolic stress test. Blood samples were collected before the exercise session, at the exercise peak, and after the end of exercise. ELISA assays were used to measure CT-1 and TNF-α plasma levels. Results:Among TA and CG, no significant differences were found for CT-1 (0.25 ± 0.14 vs 0.20 ± 0.14 fm/l; p = 0.29) and TNF-α (10.8 ± 2.7 vs 9.7 ± 4.0 pm/l; p = 0.29) basal levels. In the TA, plasma levels of CT-1 were significantly different at rest and during exercise (basal 0.25 ± 0.13 pm/l; peak 1.07 ± 1.5 pm/l; post-exercise 0.67 ± 0.77 pm/l; p = 0.04). Conversely, no significant differences were found between basal, peak and post-exercise plasma values of TNF-α (basal 10.8 ± 2.7 pm/l; peak 11.7 ± 2.1 pm/l; post-exercise 11.4 ± 2.5 pm/l; p = 0.78) in TA. Conclusions: This study gives novel insights on the behavior of inflammatory cytokines during physical exercise in athletes and healthy individuals.     

Role of proinflammatory markers and NT-proBNP in patients with an implantable cardioverter-defibrillator and an electrical storm

BackgroundSeveral studies have attempted to identify risk factors for the development of an electrical storm (ES), which is defined as ⩾3 separate ventricular tachyarrhythmic (VT/VF) events, but in the majority of studies no triggers have been found. However, little is known about the role of inflammation and NT-proBNP in patients with ES. The aim of this study was therefore to assess the relationship of Interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP) and NT-proBNP serum concentrations in ICD-patients with or without single spontaneous ventricular tachyarrhythmic events (VT/VF) and in ES.MethodsMarkers were determined in 51 patients without ICD-intervention, in 15 ICD-patients with single VT/VF-episodes during 9-months follow-up and in 20 ICD-patients with ES (blood sampling performed within 60 min after fulfilling ES criteria). VT/VF-episodes were analysed by stored ICD-electrograms.ResultsAll patients had idiopathic dilated cardiomyopathy (n = 23) or coronary artery disease (n = 63). Patients with ES revealed significantly higher mean serum concentrations of all markers (IL-6 15.19 ± 10.34 pg/mL, hs-CRP 20.12 ± 14.4 mg/L, NT-proBNP 4799 ± 4596 pg/mL) compared to baseline values of patients with single VT/VF-events during follow-up (IL-6 8.37 ± 5.8 pg/mL (p = 0.03), hs-CRP 4.7 ± 5.3 mg/dL (p < 0.001), NT-proBNP 1913 ± 2665 pg/mL (p = 0.04)) and compared to baseline values of ICD-patients without device intervention (IL-6 4.62 ± 3.66 pg/mL (p < 0.001), hs-CRP 4.1 ± 3.4 mg/L (p < 0.001), NT-proBNP 1461 ± 2281 pg/mL (p < 0.001)). In 9/20 patients presenting with ES (45%) baseline values were available. All markers were significantly higher during ES compared to event-free determination (IL-6 14.54 ± 10.43 vs. 7.03 ± 2.83 pg/mL (p = 0.04), hs-CRP 19.07 ± 16.07 vs. 6.5 ± 3.9 mg/L (p = 0.02), NT-proBNP 4218 ± 2561 vs. 2099 ± 1279 pg/mL (p = 0.03)).ConclusionsElectrical storm is associated with significantly elevated IL-6, hs-CRP and NT-proBNP serum concentrations in ICD-patients with structural heart disease. Thus, ES may be triggered by proinflammatory activity. Combined intraindividual elevation of determined markers might help to identify patients at risk of impending electrical storm.     

The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen

The aim of this study was to determine the effects of antioxidants such as reduced glutathione (GSH) and cysteine in Laiciphose® extender on semen parameters, fertilizing ability, lipid peroxidation (LPO) level and glutathione peroxidise (GPx) activity of post-thawed bull semen. Totally 54 ejaculates of three bulls were used in the study. Five groups, namely; GSH (0.5 and 2 mM), cysteine (5 and 10 mM) and control group, were conducted to test the antioxidants in Laiciphose®. Insemination doses were processed that each 0.25-mL straw contained 15 × 10⁶ sperm. The addition of antioxidants did not present any significant effect on the percentages of post-thaw sperm morphology (acrosome and total abnormalities), subjective, CASA and progressive motilities, as well as sperm motility characteristics (VAP, VSL, VCL, LIN and ALH), compared to the control groups (P > 0.05). GSH 0.5 mM (55.5 ± 7.38%) and cysteine 10 mM (48 ± 5.65%) led to lower rates of DNA damage, compared to control (P < 0.05). As regards to MDA level, cysteine at 10 mM dose gave the highest level (4.99 ± 0.44 nmol/L) (P < 0.001). GPx activity was demonstrated to be higher level upon the addition of 5 mM cysteine when compared to the other groups (P < 0.05). With respect to fertility results based on 60-day non-returns, the supplementation of antioxidants did not present significant differences (P > 0.05). The results of this study may provide an useful information for the future studies in this area. So, further studies could be suggested to achieve better information in terms of the DNA damage and fertilizing capacity of bull sperm frozen with effective antioxidants.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Synthesis of highly potent novel anti-tubercular isoniazid analogues with preliminary pharmacokinetic evaluation

Thirty two novel isoniazid analogues were prepared by one-pot three component condensations of isoniazid (INH), 3-mercaptopropionic acid and various aryl/heteroaryl aldehydes. The synthesized compounds were evaluated for their anti-TB activity against Mycobacterium tuberculosis H37Rv (MTB) and cytotoxicity. Among the compounds, compound N-(2-(4-(benzyloxy) phenyl)-4-oxo-1,3-thiazinan-3-yl) isonicotinamide (17) inhibited MTB with MIC of 0.12 μM and was three times more potent than INH. The main pharmacokinetic parameters after intravenous administration (10 mg/kg body weight) in male Wistar rats viz. t_½, K_el, mean plasma clearance and mean volume of distribution were found to be 1.14 ± 0.20 h, 0.62 ± 0.10 h^?1, 22.48 ± 0.16 mL/kg/min and 1.99 ± 0.49 L, respectively. The systemic absorption was slow after oral administration (50 mg/kg body weight). The peak plasma concentration was found to be 1.31 ± 0.06 μg/mL attained in 3 h. The bioavailability was found to be 16.7%.     

Structural insights into the dual substrate specificities of mammalian and Dictyostelium dihydropteridine reductases toward two stereoisomers of quinonoid dihydrobiopterin

Up to now, d-threo-tetrahydrobiopterin (DH₄, dictyopterin) was detected only in Dictyostelium discoideum, while the isomer l-erythro-tetrahydrobioterin (BH₄) is common in mammals. To elucidate the mechanism of DH₄ regeneration by D. discoideum dihydropteridine reductase (DicDHPR), we have determined the crystal structure of DicDHPR complexed with NAD⁺ at 2.16 Å resolution. Significant structural differences from mammalian DHPRs are found around the coenzyme binding site, resulting in a higher K_m value for NADH (K_m = 46.51 ± 0.4 μM) than mammals. In addition, we have found that rat DHPR as well as DicDHPR could bind to both substrates quinonoid-BH₂ and quinonoid-DH₂ by docking calculations and have confirmed their catalytic activity by in vitro assay.Structured summary of protein interactionsDHPR binds to DHPR by X-ray crystallography (View interaction)     

pH-Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain

Formation of spider silk from its constituent proteins—spidroins—involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild‐type (wt) E. australis NT at pH 7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5–6 Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH 7 and pH 6 and indicate that the biological function of NT depends on conversion between the two conformations.     

Epitope analysis of white spot syndrome virus of Penaeus monodon by in vivo neutralization assay employing a panel of monoclonal antibodies

A panel of six monoclonal antibodies (MAbs) against the major envelope proteins VP18, VP26 and VP28 of white spot syndrome virus (WSSV) was evaluated for neutralization of the virus in vivo in Penaeus monodon. WSSV stock diluted to 1 × 10^?6 resulting in 100% mortality on 12 day post injection (dpi) was used as optimum infectious dose of virus for challenge. Constant quantity (100 μg/ml) of MAbs C-5, C-14, C-33, C-38, C-56 and C-72 was incubated separately with WSSV (1 × 10^?6 dilution) at 27 °C for 90 min and injected to shrimp. WSSV infection was neutralized by the MAbs C-5, C-14 and C-33 with a relative percent survival (RPS) of 60, 80 and 60 on 12 dpi, respectively compared to 100% mortality in positive control injected with WSSV alone. MAbs C-38, C-56 and C-72 could neutralize WSSV infection with RPS on 12 dpi of 40, 30 and 30, respectively. Shrimp injected with WSSV (1 × 10^?6 dilution) incubated with panel of the MAbs at 100 μg/ml separately were subjected to nested PCR analysis at 0, 8, 12, 24, 36, 48 and 72 hour post injection (hpi) to provide further evidence for neutralization. MAbs C-5, C-14 and C-33 showed delay in WSSV positivity by 24 and 48 hpi by 2nd and 1st step PCR, respectively. MAbs C-38, C-56 and C-72 showed WSSV positivity by 12 and 24 hpi by 2nd and 1st step PCR, respectively. Shrimp injected with WSSV alone showed WSSV positivity by 8 and 12 hpi by 2nd and 1st step PCR, respectively. The study clearly shows that infectivity of WSSV could be delayed by MAbs C-14, C-5 and C-33.     

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aimsMesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.MethodsMSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.ResultsPost-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.ConclusionsA method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.     

Crystal structure of human MTH1 and the 8-oxo-dGMP product complex

MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9 Å) and its complex with the product 8-oxo-dGMP (1.8 Å). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design.