C. A. Smith

Effect of glycation on basic fibroblast growth factor induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: Possible relevance to wound healing in diabetes

Ineffectual wound healing in hyperglycaemic patients suffering from diabetes mellitus is characterised by a reduction in capillary reformation (angiogenesis). Basic fibroblast growth factor (FGF-2) is secreted by fibroblasts, macrophages and in particular endothelial cells (EC) in response to tissue injury and is important in promotion of neovascularisation. Recently, glycation of FGF-2 has been shown to significantly reduce its activity in vitro. We have examined the kinetics of FGF-2 glycation and compared its ability with that of native FGF-2 to activate mitogenesis, capillary formation and associated signal transduction in bovine aortic EC (BAEC). FGF-2 was exposed to 0.25 M glucose-6-phosphate (G-6-P) for 24–72 h and the degree of glycation determined by matrix assisted laser desorption ionisation mass spectrometry. Native FGF-2 was heterogeneous with Mw in the range 15,153.6–17,903 Da. After 24 h incubation with G-6-P there was evidence of glycation, and the mass increase corresponded to addition of 2.7 mol of G-6-P residues; after 48 h, 4 mol sugar was added and this increased to 8.7 after 72 h. Dimerisation of FGF-2 was observed after 72 h of treatment. Induction of mitogenesis in BAEC was significantly reduced by 25%–40% after treatment for 48–96 h with glycated (24 h) FGF-2 (gFGF-2;100 pg/ml–5 ng/ml; P < 0.05), whilst capillary tubule formation was significantly reduced by between 60% and 90% (100 pg/ml–1 ng/ml; P < 0.05) after 5 days compared to native FGF-2. Subsequent investigation of the signal transduction molecules associated with mitogenesis showed a reduction in FGF-2 induced tyrosine phosphorylated proteins of approximate Mw 20–150 kDa between 10 min and 24 h, in particular, mitogen activated protein kinase (MAPK)/early response kinase (ERK-1, ERK-2), after glycation. To determine the reason for reduced angiogenic activity of gFGF-2, we compared its binding characteristics to that of native FGF-2. Total binding of gFGF-2 to the cell surface was significantly reduced in BAEC analysed by FACS compared to native FGF-2 (P < 0.05). Further investigation using 125I-labelled differentially washed samples, demonstrated a significant reduction in gFGF-2 binding to the high affinity tyrosine kinase receptor (46%) compared to native FGF-2. In summary, glycation of FGF-2 in vitro occurs rapidly within 24 h in the presence of elevated levels of G-6-P. Glycation caused a significant reduction in the ability of FGF-2 to bind to the tyrosine kinase receptor and activate signal transduction pathways responsible for both mitogenesis and capillary formation in BAEC. These results could help to explain the mechanism behind impaired wound healing in patients with diabetes mellitus.

Aminosalicylic acid reduces the antiproliferative effect of hyperglycaemia, advanced glycation endproducts and glycated basic fibroblast growth factor in cultured bovine aortic endothelial cells: Comparison with aminoguanidine

Hyperglycaemia reduces proliferation of bovine aortic endothelial cells in vitro. A similar effect in vivo may contribute to longterm complications of diabetes such as impaired wound-healing and retinopathy.

Molecular biology and biotechnology

Molecular biology and biotechnology , Molecular biology and biotechnology , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

The Effect of Temperature on Viability of Carbon- and Nitrogen-Starved Escherichia coli

Escherichia coli was grown in a defined medium at optimum temperature and then transferred to each of five different starvation regimes at 5°C, 20°C, or 37°C, for 1000 hours. Cells were maintained with growth-limiting amounts of carbon or nitrogen, or without either or both nutrients. Bacterial cell viability was assessed by dilution plating, the reduction of 2(p-indophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT), direct viable counts (DVC), and microcolony development. The recoverability of cells on solid medium declined most rapidly, and to the greatest extent in most cases, in cultures maintained at 37°C. Only nitrogen-starved cells maintained at 5°C became completely nonculturable. The reduction of INT consistently indicated higher numbers of viable cells compared to the other methods in all cultures. The viabilities of carbon- and nitrogen-limited cells, assessed by all methods, were similar to one another at each of the temperatures. Viability was lowest at 37°C. Nutrient-downshifted cells also followed a temperature-dependent pattern of survival with viability lowest at 37°C. Morphological differences were noted at different temperatures but were most obvious for nitrogen-starved cells at 37°C, which increased in length.

A semi-micro quantitative assay for determination of chitinolytic activity in microorganisms

Abstract A semi-micro quantitative assay for microbial chitinolytic activity has been developed. The amount of dye released from a chitin-azure substrate by chitinase producing organisms was determined densitometrically and used as a measure of chitinolytic activity. This activity could be expressed as units of chitinase·mg −1 of inoculum protein, after calibration of the system. A linear relationship was found between dye release and inoculum protein content in constitutive and induced microorganisms. The assay described offers advantages over existing techniques. It is simple, quick and suitable for use in screening programmes or in studies on enzyme kinetics.

Energy in biological systems

1 Energy and life.- 1.1 Introduction.- 1.2 Thermodynamics.- 1.3 Free energy, enthalpy and entropy.- 1.4 The biochemistry of adenosine triphosphate (ATP).- 1.5 Overview.- Answers to exercises.- Questions.- 2 An overview of bioenergetics.- 2.1 Introduction.- 2.2 Organization of metabolism.- 2.3 What is catabolism for?.- 2.4 Acquiring energy from the environment.- 2.5 Strategies for generating ATP and NADPH in catabolism.- 2.6 Overview.- Answers to exercises.- Questions.- 3 Electron transport.- 3.1 Introduction.- 3.2 Electron transport.- 3.3 Oxidative phosphorylation.- 3.4 Synthesis of ATP.- 3.5 Photosynthesis.- 3.6 The mechanism of ATP synthesis.- 3.7 Overview.- Answers to exercises.- Questions.- 4 The tricarboxylic acid cycle.- 4.1 Introduction.- 4.2 How the cycle was elucidated.- 4.3 Some biochemical details.- 4.4 The TCA cycle in relation to other cellular processes.- 4.5 Overview.- Answers to exercises.- Questions.- 5 Glycolysis.- 5.1 Introduction.- 5.2 Early history of the study of glucose metabolism.- 5.3 The Embden-Meyerhof pathway.- 5.4 Biological significance of the glycolytic pathway.- 5.5 Glycogen and other polysaccharides.- 5.6 Other monosaccharides.- 5.7 The pentose phosphate pathway.- 5.8 Overview.- Answers to exercises.- Questions.- 6 Lipids: breakdown of fatty acids brown fat ruminant metabolism.- 6.1 Introduction.- 6.2 Triacylglycerols as energy reserves.- 6.3 Oxidation of fatty acids.- 6.4 Thermogenesis.- 6.5 Ruminant metabolism.- 6.6 Overview.- Answers to exercises.- Questions.- 7 Amino acid catabolism.- 7.1 Introduction.- 7.2 Protein degradation.- 7.3 Removal of nitrogen from amino acids.- 7.4 Essential amino acids.- 7.5 Carbon chain catabolism.- 7.6 Interconversion of amino acids and transamination.- 7.7 Pathways for nitrogen excretion.- 7.8 Biosynthesis of urea.- 7.9 Overview.- Answers to exercises.- Questions.- Answers to questions.

Specific activities of glycosyltransferase enzymes vary with monoclonal antibody productivity in murine hybridomas

SummaryTwo hybridomas (PQXB1/2 and PQXB2/2) derived from the same parental cell lines showed a three times difference in specific antibody productivity. Measurement of the specific activities of three glycosyltransferase enzymes (galactosyl-, sialyl- and fucosyl-) showed significantly higher values (up to x12) in the high secretor (PQXB1/2). These results infer that high glycosylatory activities are associated with effective monoclonal antibody secretion.

Statistical Methods in Biology.

Preface 1. Introduction 2. Variability and frequency distributions 3. Estimation, standard errors and confidence limits 4. The basic idea of a significance test 5. Simple significance tests based on the normal distribution 6. The use of t-tests for small smaples 7. Contingency tables and X2 8. X2-tests of goodness-of-fit and homogeneity 9. The correlation of measurements 10. Regression analysis 11. Simple experimental design and the analysis of variance 12. Introduction to factorial experiments 13. Random samples and random numbers 14. Partial correlation and multiple regression 15. Non-parametric and distribution-free tests 16. Notes on numerical calculation, calculators and computers Appendix tables Index.

Computer applications in the biomolecular sciences. Part 1: molecular modelling

This article describes the basic tenets of molecular modelling, a computer-based means of visualizing and investigating the structures and properties of molecules. Its emphasis is on the applications of molecular modelling to the study of biological molecules and its uses in teaching students in the life sciences.

The eukaryotic chromosome

CONTENTS INTRODUCTION .......................................................... 151 CHEMICAL COMPOSITION OF METAPHASE CHROMOSOMES ........... ] ........ 152 TITRATION AND MAGNESIUM ION ]~INDING TO METAPHASE CHROMOSOMES ... 158 CHROMATID SUBUNITS AND STRANDEDNESS ................................ 159 STRUCTURE OF THE METAPHASE CHROMOSOME ............................ 160 THE REPLICATION UNIT OF THE ~¢~AMMALIAN CHROMOSOME ................ 16~. ATTACHMENT OF THE CHROMOSOME TO THE NUCLEAR MEMBRANE ............ 162 ANALOGIES IN THE CONTROL SYSTEMS (~F PROKARYOTES AND~.UKARYOTES .... 163 COARSE CONTROL ........................................................ 166 FINE CONTROL .......................................................... 173