Mechanism of hydrolysis of dicholine esters with long polymethylene chain by human butyrylcholinesterase

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine (n=4) and sebacyldicholine (n=8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.     

Effects of an Agropyron chromosome on endosperm proteins in common wheat (Triticum aestivum L.)

An Agropyron chromosome having a gene conferring blue color on the aleurone layer of the kernel endosperm causes a 15% increase in total grain protein content when it is added to the common wheat (2n=42) complement. In contrast, there is no effect of this chromosome on total protein content if it replaced part of a wheat chromosome. Endosperm protein components of isolines having blue aleurone due to the Agropyron chromosome being added (2n=44) or translocated (2n=42) were compared to normal nonblue isoline counterparts. Gliadin proteins separated by aluminum lactate (pH 3.2) polyacrylamide gel electrophoresis (PAGE) in one or two dimensions showed greater staining intensity for the blue addition isolines (2n=44) than nonblue (2n=42) isolines. However, the 42-chromosome blue isoline did not show increased protein staining over the nonblue isoline, but at least five protein differences were detected between the lines. SDS-PAGE showed that blue and nonblue differences were expressed primarily in the gliadins, but also in the glutenin, globulin, and albumin proteins.This research was supported by a D. F. Jones Postdoctoral Fellowship to K. M. Soliman and by Western Regional Project W-132, Genotype-environment interactions related to end-product uses in small grains.     

BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

Celiac sprue: correlation with murine T cell responses to wheat gliadin components

Celiac sprue is a disease in humans that is characterized by small intestinal mucosal injury and malabsorption. Dietary exposure to gliadin and similar proteins in rye and barley activates the disease in susceptible individuals. Celiac sprue appears to be the only disease with a marked HLA-association in which the proteins that activate the disease currently are well known. However, bread wheat gliadins are a complex mixture of proteins that contain at least 40 different components. In the present study we have purified the major gliadin components of Scout 66 wheat and used these proteins to examine murine T cell proliferative responses to gliadin. Differences in T cell proliferation stimulated by alpha-, beta-, gamma-, and omega-gliadins paralleled the known structural differences among these proteins. After priming with whole gliadin, the components that stimulated T cell proliferation were the same as those recognized to activate celiac sprue in humans. Studies with reduced and alkylated A-gliadin (i.e., S-methyl A-gliadin) suggested that epitopes determined by the native conformation of A-gliadin may be important in its interaction with T cells. By using three different A-gliadin peptides that span the entire molecule, T cell proliferative responses were shown to be stimulated predominantly by antigenic determinants on the NH2-terminal peptide.     

Passive surveillance of human African trypanosomiasis in Côte d’Ivoire: Understanding prevalence,clinical symptoms and signs,and diagnostic test characteristics

BackgroundLittle is known about the diagnostic performance of rapid diagnostic tests (RDTs) for passive screening of human African trypanosomiasis (HAT) in Côte d’Ivoire. We determined HAT prevalence among clinical suspects, identified clinical symptoms and signs associated with HAT RDT positivity, and assessed the diagnostic tests’ specificity, positive predictive value and agreement.MethodsClinical suspects were screened with SD Bioline HAT, HAT Sero-K-Set and rHAT Sero-Strip. Seropositives were parasitologically examined, and their dried blood spots tested in trypanolysis, ELISA/Tbg, m18S-qPCR and LAMP. The HAT prevalence in the study population was calculated based on RDT positivity followed by parasitological confirmation. The association between clinical symptoms and signs and RDT positivity was determined using multivariable logistic regression. The tests’ Positive Predictive Value (PPV), specificity and agreement were determined.ResultsOver 29 months, 3433 clinical suspects were tested. The RDT positivity rate was 2.83%, HAT prevalence 0.06%. Individuals with sleep disturbances (p<0.001), motor disorders (p = 0.002), convulsions (p = 0.02), severe weight loss (p = 0.02) or psychiatric problems (p = 0.04) had an increased odds (odds ratios 1.7–4.6) of being HAT RDT seropositive. Specificities ranged between 97.8%-99.6% for individual RDTs, and 93.3–98.9% for subsequent tests on dried blood spots. The PPV of the individual RDTs was below 14.3% (CI 2–43), increased to 33.3% (CI 4–78) for serial RDT combinations, and reached 67% for LAMP and ELISA/Tbg on RDT positives. Agreement between diagnostic tests was poor to moderate (Kappa ≤ 0.60), except for LAMP and ELISA/Tbg (Kappa = 0.66).ConclusionIdentification of five key clinical symptoms and signs may simplify referral for HAT RDT screening. The results confirm the appropriateness of the diagnostic algorithm presently applied, with screening by SD Bioline HAT or HAT Sero-K-Set, supplemented with trypanolysis. ELISA/Tbg could replace trypanolysis and is simpler to perform.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03356665","term_id":"NCT03356665"}}NCT03356665.     

Novel water-soluble near-infrared cyanine dyes: synthesis, spectral properties, and use in the preparation of internally quenched fluorescent probes

In this paper, we describe the synthesis and the photophysical properties of two novel near-infrared (NIR) cyanine dyes (NIR5.5-2 and NIR7.0-2) which are water soluble potential substitutes of the commercially available Cy 5.5 and Cy 7.0 fluorescent labels respectively. For each one of these cyanine dyes, the synthetic strategy relies on the postsynthetic derivatization of a cyanine precursor in order to introduce the key functionalities required for bioconjugation of these NIR fluorophores. For NIR5.5-2, a reactive amino group was acylated with an original trisulfonated linker for water solubility. For NIR7.0-2, a vinylic chlorine atom was derivatized through a SRN1 reaction for the introduction of a monoreactive carboxyl group for labeling purposes. Unexpectedly, when these two fluorophores were closely associated within a peptidic architecture, mutual fluorescence quenching between NIR5.5-2 and NIR7.0-2 was observed both at 705 (NIR5.5-2) and 798 nm (NIR7.0-2). On the basis of this property, a novel internally quenched caspase-3-sensitive NIR fluorescent probe was prepared.     

Effects of electromyostimulation training on muscle strength and power of elite rugby players

The present study investigated the influence of a 12-week electromyostimulation (EMS) training program performed by elite rugby players. Twenty-five rugby players participated in the study, 15 in an electrostimulated group and the remaining 10 in a control group. EMS was conducted on the knee extensor, plantar flexor, and gluteus muscles. During the first 6 weeks, training sessions were carried out 3 times a week and during the last 6 weeks, once a week. Isokinetic torque of the knee extensors was determined at different eccentric and concentric angular velocities ranging from -120 to 360 degrees .s(-1). Scrummaging and full squat strength, vertical jump height and sprint-running times were also evaluated. After the first 6 weeks of EMS, only the squat strength was significantly improved (+8.3 +/- 6.5%; p < 0.01). After the 12th week, the -120 degrees .s(-1) maximal eccentric, 120 and 240 degrees .s(-1) maximal concentric torque (p < 0.05), squat strength (+15.0 +/- 8.0%; p < 0.001), squat jump (+10.0 +/- 9.5%; p < 0.01), and drop jump from a 40-cm height (+6.6 +/- 6.1%; p < 0.05) were significantly improved. No significant change was observed for the control group. A 12-week EMS training program demonstrated beneficial effects on muscle strength and power in elite rugby players on particular tests. However, rugby skills such as scrummaging and sprinting were not enhanced.     

Multiple congenital malformations including generalized hypertrichosis with gum hypertrophy in a child exposed to valproic acid in utero

Fetal valproate syndrome results from in utero exposure to valproic acid. It is characterized by a distinctive facial appearence, a cluster of minor and major anomalies, and central nervous system dysfunction. We report on a child exposed prenatally to valproic acid with unusual anomalies. This patient was the first child of young parents. Mother had several generalized seizures one year before this pregnancy, and since than she took valproic acid. Pregnancy was otherwise uneventful. At birth physical examination showed generalized hypertrichosis sparing palms and soles, coarse face, gum hypertrophy, hypotonia, club feet and club hands, two annular constrictions of the right lower leg, and abnormal dermatoglyphics. Skeletal X-rays were normal. Gum hypertrophy and hypertrichosis may be part of a broader pattern of altered morphogenesis in fetus exposed to valproic acid or this patient had two conditions, fetal valproate syndrome and hypertrichosis with gum fibromatosis.     

Formation and characterization of soluble complexes of histone H1 with supercoiled DNA

We have analyzed the interaction of rat liver histone H1 with superhelical DNA. Depending on the ratio of H1 to DNA and the concentration of salt, two different types of complexes were found. Above a critical ratio of H1 to DNA, called the aggregation point, large aggregates are formed, which have a cable-like appearance in the electron microscope. Below the aggregation point, individual soluble complexes are formed, which are the subject of this study. With increasing ionic strength, the aggregation point is shifted towards lower ratios of H1 to DNA. In the soluble complexes, H1 appears to bind along superhelically intertwined DNA strands, forming a polymer. Partial digestion of the complexes with protease suggests protection of the N-terminal tail and the globular domain of H1. Similar soluble complexes were observed with various H1 fragments but not with the core histones. In the soluble complexes, similar regions of the H1 molecule are considered to be protected from cleavage by protease, as in chromatin. Therefore, these complexes appear to be a valuable model for the interaction of H1 in chromatin fibers.