Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor

Exploitation of soluble extracellular domains (ECDs) of the nicotinic acetylcholine receptor may provide a route to crystallographic studies aimed at exploring the structure and function of the intact receptor. The first step towards this goal is to manufacture and isolate soluble fragments that fold and assemble to form a functionally relevant complex. The baculovirus insect cell expression system was used to co-express soluble ECDs of all four muscle-type nicotinic acetylcholine receptor subunits (alpha, beta, gamma & delta-ECD) from Torpedo. Protein complexes were purified using either the conformationally sensitive monoclonal antibody mAb35, specific for a folded alpha subunit, or a NiNTA affinity resin, specific for a polyhistidine tag engineered on the delta-ECD. Western blotting with subunit specific antibodies confirmed the co-expression of each ECD and furthermore, indicated that the alpha, beta and gamma-ECDs were being co-purified with the polyhistidine-tagged delta-ECD. Chemical cross-linking was used to show that these co-purified proteins had indeed interacted specifically to form soluble oligomeric complexes. A low-resolution, three-dimensional image of these purified complexes, composed only of ECDs, was obtained by electron microscopy. They were shown to resemble the extracellular vestibule of the native receptor, having the same pseudo-pentameric symmetry, size and shape. Expression of incomplete sets of the four nicotinic acetylcholine receptor ECDs did not yield detectable complexes.     

The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission

Communication in the nervous system takes place at chemical and electrical synapses, where neurotransmitter-gated ion channels, such as the nicotinic acetylcholine (ACh) receptor, and gap junction channels control propagation of electrical signals from one cell to the next. Newly developed electron crystallographic methods have revealed the structures of these channels trapped in open as well as closed states, suggesting how they work. The ACh receptor has large vestibules extending from the membrane which shape the ACh-binding pockets and facilitate selective transport of cations across a narrow membrane-spanning pore. When ACh enters the pockets it triggers a concerted conformational change that opens the pore by destabilizing a gate in the middle of the membrane made by a ring of pore-lining alpha-helical segmets. The alternative 'open' configuration of pore-lining segments reshapes the lumen and creates new surfaces, allowing the ions to pass through. The gap junction channel uses a similar structural mechanism, involving coordinated rearrangements of alpha-helical segments in the plane of the membrane, to open its pore.     

Effect of temperature on seed production in the invasive grass Glyceria maxima (Hartm.) Holmb. (Poaceae) in South Africa

Temperature is one of the main factors that determine sexual reproduction in terrestrial and emergent aquatic plant species. The effect of temperature on sexual reproduction and seed production of Glyceria maxima (Hartm.) Holmb. in the southern hemisphere is unknown. Glyceria maxima collections in February 2010 at three isolated infestations in KwaZulu-Natal failed to yield a single seed, only empty panicles. Laboratory experiments showed that vernalisation had no consistent effect on seed production. Field- and laboratory-grown plants produced seeds in the 2010/2011 season, because of having sufficient time at optimum temperatures required for seed production (1 491 and 1 585 hours, respectively), compared to a shorter period (1 352 hours) of suitable temperatures during the 2009/2010 growing season. An inadequate period of optimum temperatures (15–25°C) during seed production resulted in the lack of seeds in the field in the 2009/2010 growing season. This study showed that temperature and duration of exposure thereto during the seed-production period play vital roles in G. maxima sexual reproduction.     

The association between familial distal renal tubular acidosis and mutations in the red cell anion exchanger (band 3, AE1) gene.

In distal renal tubular acidosis (dRTA) the tubular secretion of hydrogen ion in the distal nephron is impaired, leading to the development of metabolic acidosis, frequently accompanied by hypokalemia, nephrocalcinosis, and metabolic bone disease. The condition can be familial, when it is usually inherited as an autosomal dominant, though there is a rarer autosomal recessive form associated with nerve deafness. It has been shown that the autosomal dominant form of dRTA is associated with a defect in the anion exchanger (AE1) of the renal collecting duct intercalated cell. This transporter is a product of the same gene (AE1) as the erythrocyte anion exchanger, band 3. In this review we will look at the evidence for this association. Studies of genomic DNA from families with this disorder have shown, both by genetic linkage studies and by DNA sequencing, that affected individuals are heterozygous for mutations in the AE1 gene whilst unaffected family members have a normal band 3 sequence. Mutations have been found in the region of proposed helices 6 and 7 of the membrane domain of band 3 and involve amino acids Arg-589 and Ser-613, and in the COOH-terminal domain of band 3. Studies of red cell band 3 from these families have provided information on the effect these mutations have on the structure and function of erythrocyte band 3. Expression studies of the erythroid and kidney isoforms of the mutant AE1 proteins, in Xenopus laevis oocytes, have shown that they retained chloride transport activity, suggesting that the disease in the dRTA families is not related simply to the anion transport activity of the mutated proteins. A possible explanation for the dominant effect of these mutant AE1 proteins in the kidney cell is that these mutations affect the targeting of AE1 from the basolateral to the apical membrane of the alpha-intercalated cell.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Female Gender Is a Social Determinant of Diabetes in the Caribbean: A Systematic Review and Meta-Analysis

BackgroundDiabetes (DM) is estimated to affect 10–15% of the adult population in the Caribbean. Preventive efforts require population wide measures to address its social determinants. We undertook a systematic review to determine current knowledge about the social distribution of diabetes, its risk factors and major complications in the Caribbean. This paper describes our findings on the distribution by gender.MethodsWe searched Medline, Embase and five databases through the Virtual Health Library, for Caribbean studies published between 2007 and 2013 that described the distribution by gender for: known risk factors for Type 2 DM, prevalence of DM, and DM control or complications. PRISMA guidance on reporting systematic reviews on health equity was followed. Only quantitative studies (n>50) were included; each was assessed for risk of bias. Meta-analyses were performed, where appropriate, on studies with a low or medium risk of bias, using random effects models.ResultsWe found 50 articles from 27 studies, yielding 118 relationships between gender and the outcomes. Women were more likely to have DM, obesity, be less physically active but less likely to smoke. In meta-analyses of good quality population-based studies odds ratios for women vs. men for DM, obesity and smoking were: 1.65 (95% CI 1.43, 1.91), 3.10 (2.43, 3.94), and 0.24 (0.17, 0.34). Three studies found men more likely to have better glycaemic control but only one achieved statistical significance.

Conclusion and Implications

Female gender is a determinant of DM prevalence in the Caribbean. In the vast majority of world regions women are at a similar or lower risk of type 2 diabetes than men, even when obesity is higher in women. Caribbean female excess of diabetes may be due to a much greater excess of risk factors in women, especially obesity. These findings have major implications for preventive policies and research.     

Mental health and well-being in primatology: Breaking the taboos

We hope to raise awareness of mental health and well-being among primatologists. With this aim in mind, we organized a workshop on mental health as part of the main program of the Winter meeting of the Primate Society of Great Britain in December 2021. The workshop was very well received. Here, we review the main issues raised in the workshop, and supplement them with our own observations, reflections, and reading. The information we gathered during the workshop reveals clear hazards to mental health and suggests that we must collectively acknowledge and better manage both the hazards themselves and our ability to cope with them if we are to avert disaster. We call on institutions and learned societies to lead in seeking solutions for the benefit of primatologists and primatology.