Tryptophan environment, secondary structure and thermal unfolding of the galactose-specific seed lectin from Dolichos lablab: fluorescence and circular dichroism spectroscopic studies

Fluorescence and circular dichroism spectroscopic studies were carried out on the galactose-specific lectin from Dolichos lablab seeds (DLL-II). The microenvironment of the tryptophan residues in the lectin under native and denaturing conditions were investigated by quenching of the intrinsic fluorescence of the protein by a neutral quencher (acrylamide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The results obtained indicate that the tryptophan residues of DLL-II are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains residing close to at least some of the tryptophan residues under the experimental conditions. Analysis of the far UV CD spectrum of DLL-II revealed that the secondary structure of the lectin consists of 57% alpha-helix, 21% beta-sheet, 7% beta-turns and 15% unordered structures. Carbohydrate binding did not significantly alter the secondary and tertiary structures of the lectin. Thermal unfolding of DLL-II, investigated by monitoring CD signals, showed a sharp transition around 75 degrees C both in the far UV region (205 nm) and the near UV region (289 nm), which shifted to ca. 77-78 degrees C in the presence of 0.1 M methyl-beta-D-galactopyranoside, indicating that ligand binding leads to a moderate stabilization of the lectin structure.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.     

C<Subscript>1</Subscript>-/C<Subscript>2</Subscript>-aromatic-imino-glyco-conjugates: experimental and computational studies of binding,inhibition and docking aspects towards glycosidases isolated from soybean and jack bean

Several C₁-imino conjugates of d-galactose, d-lactose and d-ribose, where the nitrogen center was substituted by the salicylidene or naphthylidene, were synthesized and characterized. Similar C₂-imino conjugates of d-glucose have also been synthesized. All the glyco-imino-conjugates, which are transition state analogues, exhibited 100% inhibition of the activity towards glycosidases extracted from soybean and jack bean meal. Among these, a galactosyl-napthyl-imine-conjugate (1c) showed 50% inhibition of the activity of pure α-mannosidase from jack bean at 22 ± 2.5 μM, and a ribosyl-naphthyl-imine-conjugate (3c) showed at 31 ± 5.5 μM and hence these conjugates are potent inhibitors of glycosidases. The kinetic studies suggested non-competitive inhibition by these conjugates. The studies are also suggestive of the involvement of aromatic, imine and carbohydrate moieties of the glyco-imino-conjugates in the effective inhibition. The binding of glyco-imino-conjugate has been established by extensive studies carried out using fluorescence emission and isothermal titration calorimetry. The conformational changes resulted in the enzyme upon interaction of these derivatives has been established by studying the fluorescence quench of the enzyme by KI as well as from the secondary structural changes noticed in CD spectra. All these studies revealed the difference in the binding strengths of the naphthylidene vs. salicylidene as well as galactosyl vs. lactosyl moieties present in these conjugates. The differential inhibition of these glyco-conjugates has been addressed by quantifying the specific interactions present between the glyco-conjugates and the enzyme by using rigid docking studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of <Emphasis Type="Italic">Dolichos lablab</Emphasis>

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively. The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Type I interferon is required for T helper (Th) 2 induction by dendritic cells

Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN‐I) in enabling this process. An IFN‐I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN‐I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1^?/? mice were incapable of initiating Th2 responses in vivo. These data demonstrate for the first time that the influence of IFN‐I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses.     

An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab

The differences among individual bile acids (BAs) in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates. Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 6 major BAs, their glycine, and taurine conjugates in mouse liver, bile, plasma, and urine was developed and validated. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for plasma and liver) was used to extract BAs. This method is valid and sensitive with a limit of quantification ranging from 10 to 40 ng/ml for the various analytes, has a large dynamic range (2500), and a short run time (20 min). Detailed BA profiles were obtained from mouse liver, plasma, bile, and urine using this method. Muricholic acid (MCA) and cholic acid (CA) taurine conjugates constituted more than 90% of BAs in liver and bile. BA concentrations in liver were about 300-fold higher than in plasma, and about 180-fold higher in bile than in liver. In summary, a reliable and simple LC-MS/MS method to quantify major BAs and their metabolites was developed and applied to quantify BAs in mouse tissues and fluids.     

Clustering protein sequences with a novel metric transformed from sequence similarity scores and sequence alignments with neural networks

Background  

The sequencing of the human genome has enabled us to access a comprehensive list of genes (both experimental and predicted) for further analysis. While a majority of the approximately 30000 known and predicted human coding genes are characterized and have been assigned at least one function, there remains a fair number of genes (about 12000) for which no annotation has been made. The recent sequencing of other genomes has provided us with a huge amount of auxiliary sequence data which could help in the characterization of the human genes. Clustering these sequences into families is one of the first steps to perform comparative studies across several genomes.     

Isometric force development, isotonic shortening, and elasticity measurements from Ca(2+)-activated ventricular muscle of the guinea pig

Isometric tension and isotonic shortening were measured at constant levels of calcium activation of varying magnitude in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs. The results were as follows: (a) The effect of creatine phosphate (CP) on peak tension and rate of shortening saturated at a CP concentration more than 10 mM; below that level tension was increased and shortening velocity decreased. We interpreted this to mean that CP above 10 mM was sufficient to buffer MgATP(2-) intracellularly. (b) The activated bundles exhibited an exponential stress-strain relationship and the series elastic properties did not vary appreciably with degree of activation or creatine phosphate level. (c) At a muscle length 20 percent beyond just taut, peak tension increased with Ca(2+) concentration over the range slightly below 10(-6) to slightly above 10(-4)M. (d) By releasing the muscle length-active tension curves were constructed. Force declined to 20 percent peak tension with a decrease in muscle length (after the recoil) of only 11 percent at 10(-4)M Ca(2+) and 6 percent at 4x10(-6)M Ca(2+). (e) The rate of shortening after a release was greater at lower loads. At identical loads (relative to maximum force at a given Ca(2+) level), velocity at a given time after the release was less at lower Ca(2+) concentrations; at 10 M(-5), velocity was 72 percent of that at 10(-4)M, and at 4x10(-6)M, active shortening was usually delayed and was 40 percent of the velocity at 10(-4) M. Thus, under the conditions of these experiments, both velocity and peak tension depend on the level of Ca(2+) activation over a similar range of Ca(2+) concentration.     

Lifestyle modifications to prevent and control hypertension. 2. Recommendations on obesity and weight loss. Canadian Hypertension Society,Canadian Coalition for High Blood Pressure Prevention and Control,Laboratory Centre for Disease Control at Health Canada,Heart and Stroke Foundation of Canada

OBJECTIVE: To provide updated, evidence-based recommendations concerning the effects of weight loss and maintenance of healthy weight on the prevention and control of hypertension in otherwise healthy adults (except pregnant women). OPTIONS: The main options are to attain and maintain a healthy body weight (body mass index [BMI] 20-25 kg/m2) or not to do so. For those at risk for hypertension, weight loss and maintenance of healthy weight may prevent the condition. For those who have hypertension, weight loss and maintenance of healthy weight may reduce or obviate the need for antihypertensive medications. OUTCOMES: The health outcome considered was change in blood pressure. Because of insufficient evidence, no economic outcomes were considered. EVIDENCE: A MEDLINE search was conducted for the years 1992-1996 with the terms hypertension and obesity in combination and antihypertensive therapy and obesity in combination. Other relevant evidence was obtained from the reference lists of the articles identified, from the personal files of the authors and through contacts with experts. The articles were reviewed, classified according to study design and graded according to level of evidence. VALUES: A high value was placed on the avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. BENEFITS, HARMS AND COSTS: Weight loss and the maintenance of healthy body weight reduces the blood pressure of both hypertensive and normotensive people. The indirect benefits of a health body weight are well known. The negative effects of weight loss are primarily the frustrations associated with attaining and maintaining a healthy weight. The costs associated with weight loss programs were not measured in the studies reviewed. RECOMMENDATIONS: (1) It is recommended that health care professionals determine weight (in kilograms), height (in metres) and BMI for all adults. (2) To reduce blood pressure in the population at large, it is recommended that Canadians attain and maintain a healthy BMI (20-25). (3) All overweight hypertensive patients (BMI greater than 25) should be advised to reduce their weight. VALIDATION: These recommendations are similar to those of the World Hypertension League, the National High Blood Pressure Education Program Working Group on Primary Prevention of Hypertension, the Canadian Hypertension Society and the Canadian Coalition for High Blood Pressure Prevention and Control. They have not been clinically tested. SPONSORS: The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at Health Canada, and the Heart and Stroke Foundation of Canada.