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     

Kidney Transplantation Is Associated with Catastrophic Out of Pocket Expenditure in India

Kidney transplantation (KT) is only viable renal replacement option for most patients in India. Most patients do not have health insurance and meet treatment expenditure from their own resources. We prospectively evaluated the expenses associated with KT and its impact on the socioeconomic status of families in a public hospital. All direct and indirect expenses incurred by the patients from the time of diagnosis of chronic kidney disease to KT were recorded. Direct expenses included physician fees, cost of drugs and disposables, dialysis, and expenses on investigations and hospitalization. Indirect expenses included travel, food, stay, and loss of income suffered by the family. Educational dropout and financial loss were also recorded. There were 43 males and 7 females between the ages of 12 and 57 years. Direct expenses ranged from US$ 2,151–23,792 and accounted for two-thirds of the total expenses. Pre-referral hospitalization, dialysis and medication accounted for majority of direct expense. Indirect expenses ranged from US$ 226–15,283. Travel expenses and loss of income accounted for most of indirect expense. About 54%, 8%, and 10% of families suffered from severe, moderate, and some financial crisis respectively. A total of 38 families had job losses, and 1 patient and 12 caregivers dropped out of studies. To conclude, KT is associated with catastrophic out-of-pocket expenditure and pushes a majority of the patients who come for treatment to public hospitals into severe financial crisis. Educational dropout and loss of jobs are other major concerns. Systematic efforts are required to address these issues.     

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.     

Parameterization and classification of the protein universe via geometric techniques

We present a scheme for the classification of 3487 non-redundant protein structures into 1207 non-hierarchical clusters by using recurring structural patterns of three to six amino acids as keys of classification. This results in several signature patterns, which seem to decide membership of a protein in a functional category. The patterns provide clues to the key residues involved in functional sites as well as in protein-protein interaction. The discovered patterns include a "glutamate double bridge" of superoxide dismutase, the functional interface of the serine protease and inhibitor, interface of homo/hetero dimers, and functional sites of several enzyme families. We use geometric invariants to decide superimposability of structural patterns. This allows the parameterization of patterns and discovery of recurring patterns via clustering. The geometric invariant-based approach eliminates the computationally explosive step of pair-wise comparison of structures. The results provide a vast resource for the biologists for experimental validation of the proposed functional sites, and for the design of synthetic enzymes, inhibitors and drugs.     

If Cell Mechanics Can Be Described by Elastic Modulus: Study of Different Models and Probes Used in Indentation Experiments

Here we investigated the question whether cells, being highly heterogeneous objects, could be described with the elastic modulus (effective Young’s modulus) in a self-consistent way. We performed a comparative analysis of the elastic modulus derived from the indentation data obtained with atomic force microscopy (AFM) on human cervical epithelial cells (both normal and cancerous). Both sharp (cone) and dull (2500-nm radius sphere) AFM probes were used. The indentation data were processed through different elastic models. The cell was approximated as a homogeneous elastic medium that had either 1), smooth hemispherical boundary (Hertz/Sneddon models) or 2), the boundary covered with a layer of glycocalyx and membrane protrusions (“brush” models). Consistency of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrated that only one model showed consistency in treating cells as a homogeneous elastic medium, namely, the brush model, when processing the indentation data collected with the dull AFM probe. The elastic modulus demonstrated strong depth dependence in all models: Hertz/Sneddon models (no brush taken into account), and when the brush model was applied to the data collected with sharp conical probes. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus, used in a self-consistent way, when using the brush model to analyze data collected with a dull AFM probe. The nature of these results is discussed.     

If Cell Mechanics Can Be Described by Elastic Modulus: Study of Different Models and Probes Used in Indentation Experiments

Here we investigated the question whether cells, being highly heterogeneous objects, could be described with the elastic modulus (effective Young’s modulus) in a self-consistent way. We performed a comparative analysis of the elastic modulus derived from the indentation data obtained with atomic force microscopy (AFM) on human cervical epithelial cells (both normal and cancerous). Both sharp (cone) and dull (2500-nm radius sphere) AFM probes were used. The indentation data were processed through different elastic models. The cell was approximated as a homogeneous elastic medium that had either 1), smooth hemispherical boundary (Hertz/Sneddon models) or 2), the boundary covered with a layer of glycocalyx and membrane protrusions (“brush” models). Consistency of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrated that only one model showed consistency in treating cells as a homogeneous elastic medium, namely, the brush model, when processing the indentation data collected with the dull AFM probe. The elastic modulus demonstrated strong depth dependence in all models: Hertz/Sneddon models (no brush taken into account), and when the brush model was applied to the data collected with sharp conical probes. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus, used in a self-consistent way, when using the brush model to analyze data collected with a dull AFM probe. The nature of these results is discussed.     

Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes

Background  

Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved β-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda.     

Large-scale chemical and chemo-enzymatic synthesis of a spacer-containing Pk-trisaccharide

The Pk-trisaccharide, linked to a solid carrier, is a potential agent for neutralization of shiga-like toxin in the gastrointestinal tract. Two approaches to the multigram-scale synthesis of a linkable Pk-trisaccharide derivative were therefore investigated. A four-step chemical synthesis yielded 8-methoxycarbonyloctyl β-lactoside in 75% yield from lactose. Further conversion of this derivative through either multistep organic synthesis or one-step enzymatic galactosylation with UDP-galactose and recombinant α-1,4-galactosyltransferase gave the Pk-trisaccharide derivative 8-methoxycarbonyloctyl α-d-galactopyranosyl-(1 → 4)-β-d-galactopyranosyl-(1 → 4)-β-d-glucopyranoside in 25% and 68% overall yields from commercial lactose, respectively.