Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Characterization of anti-tubular basement membrane antibodies in rats

Autoimmune tubulointerstitial nephritis was induced in Brown-Norway (BN) rats by immunization with bovine (Bov) tubular basement membrane (TBM) in complete Freund's adjuvant. Serum antibodies thus produced reacted to a greater extent with Bov than BN TBM antigens by indirect immunofluorescence and by radioimmunoassay with particulate (P) and collagenase-solubilized (CS) TBM. The quantities of antibodies reactive with CS TBM correlated with the intensity of tubulointerstitial pathologic changes. Antibodies eluted from kidneys reactive with BN TBM by indirect immunofluorescence were 508 times more concentrated in the kidney than in the serum, compared with 15 times for Bov TBM-reactive antibodies. The reactivity of eluted antibodies to P BN TBM was inhibited by 70% after absorption with BN CS TBM. A major CS TBM antigen of 42,000 m.w. was identified by polyacrylamide gel electrophoresis. This antigen was present in both Bov and BN TBM, and may be important in triggering autoantibody formation in this model. Lewis rats immunized under the same conditions produced antibodies reactive with BN TBM by immunofluorescence but failed to develop immune deposits in TBM of their own kidneys. Analysis of serum anti-TBM antibodies in Lewis rats revealed a selective lack of reactivity with either homologous or autologous CS TBM. These results suggest that the ability to make an immune response to one or more elements of CS TBM plays a major role in the development of autoimmune tubulointerstitial nephritis in rats.     

Studies on lipogenesis in vivo: Comparison of cholesterol and fatty acid synthesis in rats and mice

1. The importance of fatty acid synthesis as a pathway for the disposal of ingested glucose has been evaluated in rats and mice given a purified diet high in glucose and low in fat. [U-¹⁴C]Glucose was either added to the diet and fed for 24hr. or given by stomach tube as a 250mg. (mice) or 1000mg. (rats) meal. The two methods of isotope administration gave similar results. 2. Under the conditions employed fatty acid synthesis appeared to be a more important pathway for glucose disposal in mice than in rats. In mice 15·3% of ingested [U-¹⁴C]glucose was converted into fatty acid and in rats the corresponding value was 8·6%. In contrast, the conversion of [U-¹⁴C]glucose into cholesterol, as a percentage of dose, was twice as high in rats as in mice. 3. The effect of 20% of corn oil in the diet on the conversion of dietary [U-¹⁴C]glucose into fat was also investigated. Mice given diets containing 1% or 20% of corn oil converted 14·6% or 7·0% respectively of dietary [U-¹⁴C]glucose into fatty acid over a 24hr. period. There was no effect of fat on the incorporation of the isotope into cholesterol. 4. In mice given diets containing 1% or 20% of corn oil approx. 10% and 2% respectively of newly synthesized fatty acids were found in the liver. Hepatic fatty acid synthesis appears to be more sensitive to dietary fat than is extrahepatic synthesis.     

Studies on lipogenesis in vivo: Fatty acid and cholesterol synthesis in hyperglycaemic-obese mice

1. Lipogenesis has been studied in intact genetically obese mice by measuring the incorporation of a single oral dose of 250mg. of [U-¹⁴C]glucose into fatty acid and cholesterol in the liver and extrahepatic tissues. Studies were also carried out with [U-¹⁴C]glucose added to the diet and fed for 24hr. With either method of isotope administration, the conversion of [U-¹⁴C]glucose into fatty acid was greatly elevated in the livers of the obese mice. In contrast, conversion of the single dose of [¹⁴C]glucose into fatty acid in extrahepatic tissues of obese mice was only half that occurring in the non-obese litter mates. When [¹⁴C]glucose was given in the diet for 24hr. the total accumulation of labelled fatty acid in extrahepatic tissues of obese mice was slightly less than in the non-obese. Uptake of labelled glucose and conversion into fatty acid in adipose tissue of the obese mice decreased with age. 2. Conversion of the single dose of [¹⁴C]glucose into liver cholesterol was comparable in obese and non-obese mice fed on a purified low-fat diet. However, obese mice given this diet for 12 weeks accumulated 1·54% of cholesterol in the liver compared with 0·29% in the non-obese litter mates. This accumulation apparently resulted from a decrease in removal of cholesterol from the liver, rather than an increased synthesis. 3. Conversion of the single dose of [¹⁴C]glucose into extrahepatic fatty acid was decreased by 18hr. starvation proportionally as much in obese as in non-obese mice. The decrease in liver fatty acid synthesis caused by starvation also was considerable in obese mice, although somewhat less marked than in the non-obese. 4. The metabolic derangements in the liver could be more fundamental to the development of the obesity than the changes seen in extrahepatic tissues.     

Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif.

We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion.     

Gonadotropin-releasing hormone in elasmobranch (electric ray, Torpedo marmorata) brain and plasma: chromatographic and immunological evidence for chicken GnRH II and novel molecular forms.

Gonadotropin-releasing hormone (GnRH) peptides in the brain, testis and plasma of an electric ray (Torpedo marmorata) were investigated by gel filtration chromatography, reverse phase high performance liquid chromatography and radioimmunoassay with region-specific antisera. In the brain, two major forms of GnRH were demonstrated. One form had identical chromatographic and immunological properties to chicken GnRH II, and the second, novel, molecular form had structural features in common with mammalian, chicken II and salmon GnRHs. A minor, early-eluting immunoreactive peak, possibly also a novel GnRH, was also evident. Immunoreactive GnRH was not detected in the testis. In the plasma, a single major early-eluting immunoreactive peak was demonstrated. This peak, identical to the minor peak observed in the brain, is likely to represent a novel form of GnRH which has immunological properties in common with mammalian, chicken II and salmon GnRHs. Immunoreactive GnRH was not detected in the plasma of species from other vertebrate classes, including rabbit, chicken, monitor lizard, clawed toad, frog, cichlid fish and lamprey. The finding of chicken GnRH II in a species of Chondrichthyes adds further support to our hypothesis that this widespread structural variant may represent an early-evolved and conserved form of GnRH. The presence of a GnRH molecular form in the plasma of the electric ray suggests that GnRH may reach target organs (pituitary and gonads) via the general circulation in some species of Chondrichthyes.     

Approaches to breeding for salinity tolerance - a case study on Porteresia coarctata

Cereals are the world's major source of food for human nutrition. Among these, rice (Oryza sativa) is the most prominent and represents the staple diet for more than two-fifths (2.4 billion) of the world's population, making it the most important food crop of the developing world (Anon., 2000a). Rice production in vast stretches of coastal areas is hampered due to high soil salinity. This is because rice is a glycophyte and it does not grow well under saline conditions. In order to increase rice production in these areas there is a need to develop rice varieties suited to saline environments. Research has shown that Porteresia coarctata, a highly salt tolerant wild relative of rice growing in estuarine soils, is an important material for transferring salt tolerant characteristics to rice. It is quite possible that Porteresia may be used as a parent for evolving better and truly salt resistant varieties. The inadequate results and the difficulties associated with conventional breeding techniques necessitate the use of the tools of crop biotechnology in unravelling some of the characteristics of Porteresia that have been highlighted in this report. In view of the limited resources available for increasing salinity tolerance to the breeders to wild rice germplasm, Porteresia is undoubtedly one of the key source species for elevating salinity tolerance in cultivated rice.     

Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides

The antimicrobial polypeptides Bac7 and Bac5 (bactenecins) are stored in the large granules of bovine neutrophils as precursor forms, or probactenecins. Maturation of probactenecins has been investigated by studying the effects of stimulus-induced degranulation on this process. Stimulation of neutrophils with PMA, which is a secretagogue for specific and large granules but not for azurophils, induces a substantial discharge of uncleaved probactenecins in the extracellular medium, as revealed by Western blot analysis. When neutrophils are exposed to opsonized bacteria in the presence of cytochalasin B, resulting in exocytosis of the content of azurophils in addition to that of specific and large granules, probactenecins are secreted and rapidly converted into the corresponding mature antimicrobial peptides. Such a conversion is prevented if serine proteases, stored in the azurophils, are inhibited by pretreatment of neutrophils with PMSF. Phagocytosis, while causing a rapid discharge of the contents of azurophil and of the large granules into phagocytic vacuoles, as indicated by immunogold electron microscopy, also induces cleavage of probactenecins into mature peptides, as revealed by Western blot analysis. We conclude that the final processing of the storage forms of bactenecins arises from their interaction with the serine protease(s) of azurophils during bacteria-induced degranulation of neutrophils.