Determination of the absolute configuration of the sugar residues of complex polysaccharides by circular dichroism spectroscopy

Circular dichroism spectroscopy is used to determine the absolute configuration of the constituent sugar residues of bacterial polysaccharides from two strains of Streptococcus sanguis which are important receptors in bacterial coaggregation in the formation of dental plaque. A high-pressure liquid chromatographic method for carbohydrate analysis of glycoproteins, glycolipids, and polysaccharides has been extended to sugar chirality determination by collection of the eluted HPLC fractions and subsequent measurement of their circular dichroism spectra. The method involves methanolysis of the polysaccharide followed by formation of O-benzoyl derivatives and HPLC on reverse-phase columns. Circular dichroism spectra of the collected derivatives in acetonitrile solution in the region 230 nm show large ellipticity bands resulting from "chiral exciton" interaction among the O-benzoyl chromophores which are sensitive to the orientation of hydroxyl groups in the parent sugars. The sensitivity of the method is in the submicrogram range for the absolute configuration of single sugar residues. The circular dichroism of the intact polysaccharide in aqueous solution shows CD bands from the amide chromophore in the region 180 to 220 nm which are sensitive to the chirality of 2-acetamido sugar residues.     

Patterns of energy storage in Pseudoboeckella poppei (Crustacea,copepoda) from two contrasting lakes on Signy Island,Antarctica

The copepod Pseudoboeckella poppei (Daday) (Calanoida, Centropagidae) was sampled from Sombre and Heywood Lakes on Signy Island, Antarctica (60° S, 45° W) between January 1984 and March 1985. Sombre Lake is clear and oligotrophic with little phytoplankton and a bottom sediment low in organic content. By contrast Heywood Lake is turbid and mesotrophic; a substantial phytoplankton develops in summer and the bottom sediments are comparatively rich in organics. Both lakes freeze over for much of the year, forcing the copepods to adopt a benthic feeding strategy over winter. Adult Pseudoboeckella feed on phytoplankton when this is available, but also on detritus, diatoms and short algal filaments stirred up from the sediment. In Heywood Lake, male copepods show a smooth seasonal trend in lipid content with lipid being synthesised in early summer and utilised in late summer and winter. The summer increase in lipid content is associated with an increase in dry weight. Female lipid contents show evidence of two peaks of egg production. In Sombre Lake both male and female copepods increase in size during summer and show a wider range of lipid contents than in Heywood Lake; it is likely that this is due to the poorer winter feeding conditions which necessitate the synthesis of a much larger store of reserves during the summer. In contrast to marine calanoid copepods, lipid stores are exclusively triacylglycerol with no trace of wax ester.     

Regulation of nicotinic acetylcholine receptor function by adenine nucleotides

1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

R-banding and nonisotopic in situ hybridization: precise localization of the human type II collagen gene (COL2A1)

Summary A new mapping system, based on nonisotopic in situ hybridization combined with fluorescent staining of replicated prometaphase R-bands, is described. Replication of the bands is achieved by treatment of thymidinesynchronized cells with bromodeoxyuridine. The human COL2A1 gene was mapped to band 12q13.11–q13.12 in this manner, to illustrate the potential of the technique for improving the precision of chromosomal mapping and physical ordering of genes.     

Growth,ammonia accumulation and glutamine synthetase activity in alfalfa (Medicago sativa L.) shoots and cell cultures treated with phosphinothricin

Summary Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa L) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10 and 20 fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27 fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.Abbreviations PPT Phosphinothricin - PAT Phosphinothricin acetyltransferase     

Characterization of antigen receptor response elements within the interleukin-2 enhancer.

T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for IL-2 activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene.     

Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone γ-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat

New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.     

Construction of transposons encoding genes for β-glucosidase,amylase and polygalacturonatetrans-eliminase fromKlebsiella oxytoca and their expression in a range of gram-negative bacteria

DNA fragments containing theKlebsiella oxytoca genes encoding -glucosidase and amylase were cloned into the kanamycin resistance transposon Tn5. Another DNA fragment containing two genes for polygalacturonatetrans-eliminase was cloned into Tn1721. These newly constructed transposons were then each transposed in vivo onto the broad-host-range plasmid pR751 and conjugally transferred to a variety of Gram-negative bacteria. These were then screened for the newly acquired phenotypes.