Active intestinal chloride secretion in human carriers of cystic fibrosis mutations: an evaluation of the hypothesis that heterozygotes have subnormal active intestinal chloride secretion

To explain the very high frequency of cystic fibrosis (CF) mutations in most populations of European descent, it has been proposed that CF heterozygotes have a survival advantage when infected with Vibrio cholerae or Escherichia coli, the toxins of which induce diarrhea by stimulation of active intestinal chloride secretion. Two assumptions underlie this hypothesis: (1) chloride conductance by the CF transmembrane conductance regulator (CFTR) is the rate-limiting step for active intestinal chloride secretion at all levels of expression, from approximately zero in patients with CF to normal levels in people who are not carriers of a mutation; and (2) heterozygotes have smaller amounts of functional intestinal CFTR than do people who are not carriers, and heterozygotes therefore secrete less chloride when exposed to secretagogues. The authors used an intestinal perfusion technique to measure in vivo basal and prostaglandin-stimulated jejunal chloride secretion in normal subjects, CF heterozygotes, and patients with CF. Patients with CF had essentially no active chloride secretion in the basal state, and secretion was not stimulated by a prostaglandin analogue. However, CF heterozygotes secreted chloride at the same rate as did people without a CF mutation. If heterozygotes are assumed to have less-than-normal intestinal CFTR function, these results mean that CFTR expression is not rate limiting for active chloride secretion in heterozygotes. The results do not support the theory that the very high frequency of CF mutations is due to a survival advantage that is conferred on heterozygotes who contract diarrheal illnesses mediated by intestinal hypersecretion of chloride.     

Translocation of amino acids in the xylem of apple (Malus domestica Borkh.) trees in spring as a consequence of both N remobilization and root uptake

Nitrogen is remobilized from storage for the growth of Malus domestica leaves each spring. Seasonal patterns of N translocation in the xylem sap as a consequence of remobilization were determined in 2-year-old 'Golden delicious' trees grafted on M9 rootstocks. The trees were grown in sand culture and (15)NH(4)(15)NO(3) at 10.4 atom% abundance supplied during August-September. The following year no further N was supplied and destructive harvests were taken during bud burst and leaf growth to determine the patterns of N remobilization together with the isolation of xylem sap for an analysis of their amino acid profiles and (15)N enrichments by GC-MS. The concentration of amino acids in the xylem sap rose following bud burst, peaked at full bloom and then fell again during petal fall and fruit set. The peak in amino acid concentration corresponded with the period when the rate of N remobilization was the fastest. The majority of labelled N was recovered in Asn, Gln + Glu and Asp demonstrating that they were being translocated as a consequence of remobilization. In a second experiment, 8-year-old trees growing in an orchard were fertilized with N either in the autumn or spring. Xylem sap samples were collected in the spring and early summer and, by comparison with the amino acid profiles recovered in trees from both treatments, Asn was identified as the main compound translocated as a consequence of both remobilization and root uptake of N, although there was evidence that root uptake of N occurred later. The data are discussed in relation to quantifying the internal cycling of N in trees.     

Structural characterization and reversal of the natural organophosphate resistance of a D-type esterase, Saccharomyces cerevisiae S-formylglutathione hydrolase

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 A resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme "acyl pocket". The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold ( k i (W197I) = 16 +/- 2 mM (-1) h (-1)), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 A); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a "D-type" esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon ( k i = 42 or 80 mM (-1) h (-1), respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.     

Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells

While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.     

Multiple selection pressures generate adherence to Bergmann's rule in a Neotropical migratory songbird

Aim Bergmann's rule, the tendency for body size to be positively correlated with latitude, is widely accepted but the mechanisms behind the patterns are still debated. Bergmann's originally conceived mechanism was based on heat conservation; other proposed mechanisms invoke phylogeny, migration distance and resource seasonality. With the goal of examining these mechanisms, we quantified morphological variation across the breeding range of a Neotropical migratory songbird, the cerulean warbler (Dendroica cerulea). Location Deciduous forests of eastern North America. Methods We sampled nine cerulean warbler populations, spanning the species’ breeding range. We captured 156 males using targeted playback and model presentation, and included 127 adult males in our analyses of morphological variation. We used an information‐theoretical approach to identify climatic variables associated with geographical variation in body size. Results Cerulean warbler body size adheres to Bergmann's rule: individuals in northern populations are larger than those in southern populations. Variation in body size is best explained by variation in dry and wet‐bulb temperature and actual evapotranspiration. Main conclusions Adherence to Bergmann's rule by the cerulean warbler appears to be linked to thermodynamics (heat conservation in the north, evaporative cooling in the south) and resource seasonality. Multiple selection pressures can interact to generate a single axis of morphological geographical variation, and even subtle fluctuations in climatic variables can exert significant selection pressures. We suggest that the influence of selection pressures on migrants might be enhanced by migratory connectivity, providing further support for the important role played by this phenomenon in the ecology, evolution and population dynamics of migratory songbirds.     

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.     

Diagnosing dementia: do we get it right?

To find out whether the diagnosis of dementia agreed with findings at necropsy a detailed assessment of 27 elderly patients (mean age 82 (range 70-94] presenting with dementia was conducted at a combined department of geriatric medicine and psychiatry for the elderly. On the basis of the results the cause of the dementia was diagnosed clinically. Neuropathological examinations were performed after death. The clinical diagnosis made during life was not supported by the findings at necropsy in 11 cases. Alzheimer''s disease was overdiagnosed in life (13 cases, of which only six were confirmed at necropsy). Although the clinical investigation was limited by availability of resources, neither cranial computed tomography nor the Hachinski score helped to distinguish between multi-infarct dementia and Alzheimer''s disease in this age group. This study confirms the value of neuropathological studies in the precise diagnosis of dementia.     

Non-structural carbohydrates and nitrogen dynamics in mediterranean sub-shrubs: an analysis of the functional role of overwintering leaves

Previous studies have led to contrasting results about the role of overwintering leaves as storage sites, which is related to leaf longevity and life-form. The aim of this study was to evaluate the functional role of the leaves of four species of Mediterranean sub-shrubs, with different leaf phenology, as sources of nitrogen (N) and non-structural carbohydrates (NSC) for shoot growth. The seasonal dynamics of the concentrations and pools of N and NSC were assessed monthly in the leaves and woody organs of each species. Overwintering and spring leaves served as N and NSC sources for shoot growth in the evergreen species analyzed, providing up to 73 % and 324 % of the N demand for spring and autumn growth, respectively. Excess autumn N was stored in woody structures which contributed to the N and NSC requirements of spring growth. In the winter deciduous species, woody organs were the main N source for spring growth, while current photosynthesis from immature brachyblasts seemed to be the main carbon (C) source. Due to their short lifespan, overwintering and spring leaves did not show several translocation processes throughout their life time, their contribution to new growth being made during senescence. The successive exchange of leaf cohorts displayed by Mediterranean sub-shrubs might serve as a mechanism to recycle N and C between consecutive cohorts as plants perform the pheno-morphological changes needed to adapt their morphology to the seasonality of their environment.     

High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.