Loop L5 acts as a conformational latch in the mitotic kinesin Eg5

All members of the kinesin superfamily of molecular motors contain an unusual structural motif consisting of an α-helix that is interrupted by a flexible loop, referred to as L5. We have examined the function of L5 in the mitotic kinesin Eg5 by combining site-directed mutagenesis of L5 with transient state kinetics, molecular dynamics simulations, and docking using cryo electron microscopy density. We find that mutation of a proline residue located at a turn within this loop profoundly slows nucleotide-induced structural changes both at the catalytic site as well as at the microtubule binding domain and the neck linker. Molecular dynamics simulations reveal that this mutation affects the dynamics not only of L5 itself but also of the switch I structural elements that sense ATP binding to the catalytic site. Our results lead us to propose that L5 regulates the rate of conformational change in key elements of the nucleotide binding site through its interactions with α3 and in so doing controls the speed of movement and force generation in kinesin motors.     

Differential involvement of Galpha16 in CC chemokine-induced stimulation of phospholipase Cbeta, ERK, and chemotaxis

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.     

Effect of neosporosis on productive and reproductive performance of dairy cattle in Costa Rica

A cross-sectional study was conducted to assess the effect of neosporosis on productive and reproductive parameters in dairy cows. Cows (n=2743) from 94 farms located in the most important dairy areas in Costa Rica were used in the study. The size of the herds ranged from 32 to 379 females (mean=110, median=125). An indirect ELISA was used to determine the serostatus of the cows towards Neospora caninum. The effect of neosporosis on milk production was analysed by a mixed linear model. In addition, the effects on calving interval (days) and calving to conception interval (days) were analysed by survival analysis. The risk of abortion in relation to N. caninum serostatus was assessed by logistic regression, with herd as a random effect. Overall, 1185 of 2743 cows (43.3%) were seropositive for Neospora. Eighty-nine of 94 (94.7%) farms were classified as Neospora-seropositive. It was estimated that cows seronegative to Neospora produced an additional 84.7L of milk (P=0.6). Serostatus did not have a significant effect on the length of the calving interval in the Cox proportional hazard survival analysis (Hazard ratio=1.2, 95% CI: 0.9, 1.4). The logistic regression model had a weak positive association between serostatus and abortion (OR 1.7, 95% CI: 0.8, 3.9), but did not show a strong association between serostatus and the number of services per conception (OR=0.95, 95% CI: 0.7, 1.3). In conclusion, there were no significant effects of Neospora serostatus on production and reproductive performance in this study.     

Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.     

Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.     

Summary from the European Meeting of WHO Collaborating Centres for Nutrition, Kraków, 23-24 June 2010

In June 2010, the WHO Regional Office for Europe, in co-operation with the WHO Collaborating Centre for Nutrition at the Department of Endocrinology, Jagiellonian University, Collegium Medicum, organized in Krakow the 1st Conference of the WHO European Collaborating Centres for Nutrition. Taking part were representatives of the WHO Regional Office for Europe Nutrition, Dr Joao Breda (Programme Manager - Nutrition, Physical Activity and Obesity Programme) and representatives of the WHO Collaborating Centres for Nutrition from Denmark, Greece, Holland, the UK and Poland, as well as representatives of co-operating institutes from Portugal, Switzerland and the UK. The main objective of the meeting was to discuss the ongoing work of each Collaborating Centre and the development of a strategic plan for the coming years in the most important areas of interest: the prevention of obesity, type 2 diabetes and other NCD, nutrition and inequalities, capacity building in nutrition as well as micronutrient deficiencies (namely iodine deficiency). A final report summarising the Network Meeting is being prepared and will be released in due course.     

Thiol-oxidant monochloramine mobilizes intracellular Ca2+ in parietal cells of rabbit gastric glands

In Helicobacter pylori-induced gastritis, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the gastric mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for Ca(2+) in the cytoplasm (fura-2 AM) or intracellular stores (mag-fura-2 AM). Conditions were adjusted to screen out contributions from metal cations such as Zn(2+), for which these reporters have affinity. Exposure to NH(2)Cl (up to 200 microM) led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), in the range of 200-400 nM above baseline levels. These alterations were prevented by pretreatment with the oxidant scavenger vitamin C or a thiol-reducing agent, dithiothreitol (DTT), which shields intracellular thiol groups from oxidation by chlorinated oxidants. Introduction of vitamin C during ongoing exposure to NH(2)Cl arrested but did not reverse accumulation of Ca(2+) in the cytoplasm. In contrast, introduction of DTT or N-acetylcysteine permitted arrest and partial reversal of the effects of NH(2)Cl. Accumulation of Ca(2+) in the cytoplasm induced by NH(2)Cl is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. Ca(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca(2+)](i). Under certain conditions, NH(2)Cl may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-NH(2)Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents.