Immunochemical studies on the interaction between synthetic glycoconjugates and alpha-L-fucosyl binding lectins

Evonymus europaea lectin precipitated with alpha DGal(1----3) beta DGal(1----4)beta DGlcNAc-bovine serum albumin (BSA), alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA, alpha LFuc(1----2)beta DGal(1----4)DGlcNAc, and alpha DGal(1----3)[alpha LFuc(1----2)]beta DGal-BSA. However, the lectin neither precipitated with alpha LFuc(1----2)-beta DGal-BSA, alpha DGal(1----3)beta DGal-BSA, or beta DGal(1----4)beta DGlcNAc-BSA nor agglutinated erythrocytes of Oh phenotype having multiple terminal beta DGal(1----4)beta DGlcNAc residues. These results indicate that the minimal structural requirement for glycoprotein precipitation or cell agglutination by the lectin includes any of the three trisaccharides (fucosylated or nonfucosylated) derived from the blood group B tetrasaccharide. The monosaccharides linked to the beta-D-galactosyl residue in the blood group B tetrasaccharide, namely, alpha-D-galactose, alpha-L-fucose, and N-acetyl-beta-D-glucosamine, participate almost equally in binding to the lectin in as much as removal of any one of these sugars reduces the inhibiting potency of the resulting trisaccharide. alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA (H type 1) and alpha LFuc(1----2)beta DGal(1----4)beta DGlcNAc (H type 2) were precipitated to the same extent. The E. europaea lectin neither precipitated alpha DGal(1----4)-beta DGal(1----4)beta DGlcNAc-BSA, Lea-BSA, Leb-BSA, or beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA nor agglutinated Oh,Lea and Oh,Leb erythrocytes, demonstrating that terminal D-galactose linked alpha-(1----4) to subterminal beta-D-galactose, or alpha-L-fucose linked to N-acetylglucosamine, prevents lectin binding. Corey-Pauling-Koltun molecular models, built on the basis of data from 1H NMR and hard-sphere exo-anomeric (HSEA) calculations provided by Lemieux and co-workers [Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S., & Rao, V. S. (1980) Can. J. Chem. 58, 631-653], show that these alpha-D-galactosyl and alpha-L-fucosyl groups act to sterically hinder lectin binding to these oligosaccharides; these observations also suggest that the lectin binds to the beta-side of these oligosaccharides. These sides, on both blood group H type 1 and blood group H type 2 oligosaccharides, provide a similar contour which can fully account for their equal reactivity with E. europaea lectin. The only difference found between Lotus and Ulex I lectins in precipitating ability was that only Lotus precipitated with beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.     

The synaptonemal complex of Meloidogyne nataliei and its relationship to that of other Meloidogyne species

The four synaptonemal complexes (SC) in Meloidogyne nataliei (n=4) have normal, tripartite morphology, although the total width of the SC is only 50 nm. Each SC is attached at both ends to the nuclear envelope and there is no bouquet formation at pachytene. Pairing of homologues is regular but not complete, as there is one region on each bivalent where the SC is not formed. This region may be the fusion point of telomeres of nonhomologous chromosomes, since it is assumed that M. nataliei has been derived from an ancestral strain (n=8) via chromosomal fusions. In each pachytene nucleus there is a nonmembranous, vacuole-like structure of unknown function located in the nucleoplasm adjacent to the nucleolus, and of approximately the same volume as the nucleolus. The fact the SC structure of M. nataliei is strikingly different from that of most Meloidogyne species suggest that M. nataliei may not belong to the same phyletic group as the genus Meloidogyne.     

Desorption chemical ionization mass spectrometry of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholines and analogues

Ammonia desorption chemical ionization of ether-linked phospholipids of the type 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet-activating factors) and a series of analogues revealed a systematic fragmentation pattern that is characteristic for these compounds. The predominant ions included the protonated molecular ion and a series of fragments derived from the molecular ion having the following nominal mass losses: MH-14, MH-42, MH-59, and MH-183. Deuterated ammonia was used to elucidate the nature of several fragments. In addition, desorption chemical ionization was used to quantitate 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-phosphocholine at the nanogram/sample level.     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station     

Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis

The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated.     

Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.     

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.     

Thymosin fraction 5 causes increased serum corticosterone in rodents in vivo

In these studies it was found that i.p. injection of thymosin fraction 5 (TF5) caused a dose-dependent increase in serum corticosterone in male Swiss Webster mice and in male Wistar rats. The maximum responses were seen at 1 and 2 hr, respectively. There was no effect on serum corticosterone in mice when Thymosin alpha 1 (a 28 amino acid peptide isolated from TF5) was injected i.p. at doses up to 100 micrograms. The steroidogenic effects of TF5 were seen only when the basal levels of serum corticosterone were low (less than 80 ng/ml). In studies in which the baseline levels in the animal colony were elevated (greater than 80 ng/ml), there were no steroidogenic effects, or they were minimal. These results suggest that some component of TF5 may influence pituitary adrenal function.