Photoinactivation of δ-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The -aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of -ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.Abbreviations ALAD -aminolevulinic acid dehydratase - FMN flavin mononucleotide - O₂ ^- superoxide - H₂O₂ hydrogen peroxide - 10₂ singlet oxygen - LA levulinic acid - PBG porphobilinogen - BSA bovine serum albumin - BME 2-mercaptoethanol - SOD superoxide dismutase - pHMB para-hydroxymercuribenzoate - DTT dithiothreitol - FAD flavin adenine dinucleotide - NADH nicotinamide adenine dinucleotide     

Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry

The 8-methoxycarbonyloctyl glycosides of GlcNAc, Gal1-4Glc, Fuc1-2Fuc1-3GalNac and Fuc1-2Gal1-3[Fuc1-4]GlcNac were converted to primary amines by reaction with neat ethylenediamine and then coupled to bovine serum albumin (BSA) using diethyl squarate as the connector. The average degree of incorporation of the sugar onto the protein, as well as the molecular weight distribution, could be conveniently determined using matrix assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry thus avoiding cumbersome structure-dependent colour-tests or analysis of cleaved ligand. The present coupling method has the advantages of proceeding under very mild conditions, yielding controlled incorporation values and can reliably be used for the coupling of very small amounts (mg) of oligosaccharide.This paper is dedicated to Sen-itiroh Hakomori on the occasion of his 65th birthday     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

A neurological integrator for the oculomotor control system

This paper describes a mathematical model of a neurological integrator that has been developed to provide the very long leakage time constant required of the intgrator in the oculomotor system. The Gaussian distribution of cell thresholds and the eye-position- related discharge of the individual cells of the integrator model, and the highly specialized short-duration, high-frequency burst required of the input, have been modeled after the single-cell behavior actually observed in the oculomotor control areas of the brain stem of an alert primate.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

Preclinical pharmacokinetics of MHAA4549A,a human monoclonal antibody to influenza A virus,and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ～2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.     

Pre-Hypertension among Young Adults (20–30 Years) in Coastal Villages of Udupi District in Southern India: An Alarming Scenario

IntroductionAccording to Joint National Committee-7 (JNC-7) guidelines, a systolic blood pressure (SBP) of 120 to 139 mm Hg and/or diastolic blood pressure (DBP) of 80 to 89 mm Hg is considered as pre-hypertension. Existing evidence suggest that the cardiovascular morbidities are increasing among pre-hypertensive individuals compared to normal.ObjectiveTo assess the magnitude and factors associated with pre-hypertension among young adults (20–30 years) in coastal villages of Udupi Taluk (an area of land with a city or town that serves as its administrative centre and usually a number of villages), Udupi District, Karnataka state, India.DesignCommunity based cross sectional studySetting6 (out of total 14) coastal villages of Udupi Taluk, Karnataka state, India.Sample1,152 young adults (age group: 20–30 years) selected by stratified random sampling in 6 coastal villages of Udupi Taluk, Karnataka state, IndiaMethodA semi structured pre-tested questionnaire was used to elicit the details on socio-demographic variables, dietary habits, tobacco use, alcohol consumption, physical activity, family history of hypertension and stress levels. Anthropometric measurements and blood pressure were recorded according to standard protocols. Serum cholesterol was measured in a sub sample of the study population. Multivariate logistic regression was applied to identify the independent correlates of pre-hypertension among young adults (20–30 years).ResultsThe prevalence of pre-hypertension in the study population was 45.2% (95%CI: 42.4–48). Multivariate logistic regression analysis revealed that age group of 25–30 years (adj OR: 4.25, 95% CI: 2.99–6.05), white collared (adj OR: 2.29, 95% CI: 1.08–4.85) and skilled occupation (adj OR: 3.24, 95% CI: 1.64–6.42), students (adj OR: 2.46, 95% CI: 1.22–4.95), using refined cooking oil (adj OR: 0.53, 95% CI: 0.29–0.95), extra salt in meals (adj OR: 2.46, 95% CI: 1.52–3.99), salty food items (adj OR: 6.99, 95% CI: 3.63–13.48), pre-obese (adj OR: 1.66, 95% CI: 1.03–2.67) and obese (adj OR: 9.16, 95% CI: 2.54, 36.4) were the significant correlates of pre-hypertension.ConclusionIn the study population, prevalence of pre-hypertension among young adults (20–30 years) was high (45.2%). Biological (age 25–30 years, pre-obesity and obesity) and behavioral (sedentary occupation, intake of extra salt in meals/salty food and not using refined cooking oil) factors were associated with pre-hypertension. Study emphasizes the need of community based screening of pre-hypertension under National Rural Health Mission. It also provides apt information for the evidence based designing of interventions for lifestyle modifications among high risk young adults in the study area.     

Potential feedback between coral presence and farmerfish collective behavior promotes coral recovery

Stable between‐group differences in collective behavior have been documented in a variety of social taxa. Here we evaluate the effects of such variation, often termed collective or colony‐level personality, on coral recovery in a tropical marine farmerfish system. Groups of the farmerfish Stegastes nigricans cultivate and defend gardens of palatable algae on coral reefs in the Indo‐Pacific. These gardens can promote the recruitment, growth, and survival of corals by providing a refuge from coral predation. Here we experimentally evaluate whether the collective response of farmerfish colonies is correlated across intruder feeding guilds – herbivores, corallivores and egg‐eating predators. Further, we evaluate if overall colony responsiveness or situation‐specific responsiveness (i.e. towards herbivores, corallivores, or egg‐eaters in particular) best predicts the growth of outplanted corals. Finally, we experimentally manipulated communities within S. nigricans gardens, adding either macroalgae or large colonies of coral, to assess if farmerfish behavior changes in response to the communities they occupy. Between‐group differences in collective responsiveness were repeatable across intruder guilds. Despite this consistency, responsiveness towards corallivores (porcupinefish and ornate butterflyfish) was a better predictor of outplanted coral growth than responsiveness towards herbivores or egg‐eaters. Adding large corals to farmerfish gardens increased farmerfish attacks towards intruders, pointing to possible positive feedback loops between their aggression towards intruders and the presence of corals whose growth they facilitate. These data provide evidence that among‐group behavioral variation could strongly influence the ecological properties of whole communities.     

Synthesis of the acceptor analog αFuc(1→2)αGal-O(CH2)7 CH3: A probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase

We report the chemical synthesis of Fuc(12)Gal-O(CH₂)₇CH₃ (1) an analog of the natural blood group (O)H disaccharide Fuc(12)Gal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog Gal(3)[Fuc(12)]Gal-O(CH₂)₇CH₃ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide Fuc(12)Gal-O(CH₂)₇CH₃ (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.