Expedient syntheses of neoglycoproteins using phase transfer catalysis and reductive amination as key reactions

Starting from peracetylated chloro- or bromo-glycosyl donors ofN-acetylneurmainic acid,N-acetylglucosamine, glucose and lactose, the correspondingp-formylphenyl glycosides were synthesized stereospecifically under phase transfer catalysed conditions at room temperature in yields of 38–67%. After Zemplén de-O-acetylation, the formyl groups were directly and chemoselectively coupled to the lysine residues of bovine serum albumin by reductive amination using sodium cyanoborohydride. The conjugation reactions were followed as a function of time and under a series of different molar ratios of the reactants to provide glycoconjugates of varying degree of antigenicities. Thus, carbohydrate protein conjugates were made readily available using essentially two key reactions.Presented in part at the 15th International Carbohydrate Symposium, Yokohama, Japan, August 12–17, 1990.     

Structure elucidation of the core octasaccharide from Citrobacter PCM 1487 with the aid of 500-MHz, two-dimensional phase-sensitive correlated, relayed-coherence transfer, double-quantum, triple-quantum filtered, and N.O.E. H-n.m.r. spectra

The main features of the primary structure of the octasaccharide, - -Glcp-(1→2)-- -Glcp-(1→2)-[- -GalpNAc- (1→3)]-- -Galp-(1→3)-- -Glcp-(1→3)-[- -Hepp-(1→7)]-- -Hepp-(1→3)-- -Hep, have been determined in the ab initio manner by ¹H-n.m.r. spectroscopy without resorting to biochemical methods of analysis. Several nontypical interresidue n.O.e. values point to a preferred solution conformation of the molecule.     

Isolation of soluble yeast beta-glucans that inhibit human monocyte phagocytosis mediated by beta-glucan receptors

The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE-cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis-inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mathematical Model of Leukemic-Cell Circulation in the Mouse

L1210 leukemic cells injected in vivo are eliminated from the blood and disintegrated in organs such as the lungs and liver. We present a compartmental model which reproduces one type of in vivo experiment, based on the so-called perfusion curves. Although the data are not complete and some are only approximated, modeling gives a consistent picture of the process.     

Responses and song pattern copying of Omega-type I-neurons in the cricket,Gryllus bimaculatus,at different prothoracic temperatures

Summary Omega-type I-neurons (ON/1) (Fig. 1A) were recorded intracellularly with the prothoracic ganglion kept at temperatures of either 8–9°, or 20–22° or 30–33 °C and the forelegs with the tympanal organs kept at ambient temperature (20–22 °C). The neurons were stimulated with synthetic calling songs (5 kHz carrier frequency) with syllable periods (SP in ms) varying between 20 and 100, presented at sound intensities between 40 and 80 dB SPL. The amplitude and duration of spikes as well as response latency decreased at higher temperatures (Figs. 1 B, 2, 6). At lower prothoracic temperatures (8–9 °C) the neuron's responses to songs with short SP (20 ms) failed to copy single syllables, or with moderate SP (40 ms) copied the syllable with low signal to noise ratio (Fig. 3). The auditory threshold of the ON/1 type neuron, when tested with the song model, was temperature-dependent. At 9° and 20 °C it was between 40 and 50 dB SPL and at 33 °C it was less than 40 dB SPL (Fig. 4). For each SP, the slope of the intensity-response function was positively correlated with temperature, however, at low prothoracic temperatures the slope was lower for songs with shorter SPs (Fig. 5). The poor copying of the syllabic structure of the songs with short SPs at low prothoracic temperatures finds a behavioral correlate because females when tested for phonotaxis on a walking compensator responded best to songs with longer SPs at a similar temperature.Abbreviations epsps excitatory postsynaptic potentials - ON/1 omega-type I-neuron - SP syllable period - SPL sound pressure level     

Polish Glomales

Spores of Acaulospora dilatata and Scutellospora dipurpurascens found in Poland are described and illustrated and their occurrence and distribution are characterized and mapped. Spores of Acaulospora dilatata from Poland do not differ from those originally described in the United States of America. The germination shield found in a number of spores is described and illustrated, and compared with that occurring in members of the genus Scutellospora. Acaulospora dilatata was found in five of the 303 soil samples taken from around the roots of Ammophila arenaria colonizing maritime sand dunes of the Sowinski National Park. Polish specimens of S. dipurpurascens are similar in size, wall structure, and reaction in Melzer's reagent to those described from the type localized in the United States of America. However, some spores from Poland have a thicker wall, greater sporogenous cells, and are somewhat darker coloured. They were recovered from 34 soils sampled from forests, gardens, sand dunes, and both cultivated and uncultivated soils. S. dipurpurascens was commonly associated with different plants of the Hel Peninsula and occurred regularly among the roots of Ammophila arenaria growing in the Slowinski National Park. Both species were found for the first time in Poland and are probably new to Europe.     

Interconversion of the salicylic acid signal and its glucoside in tobacco

Salicylic acid (SA) has been proposed to play a role in the induction of pathogenesis-related (PR) proteins and systemic acquired resistance (SAR) in tobacco. Since SA is rapidly converted to salicylic acid β-glucoside (SAG) in tobacco, we have attempted to assess the role of SAG in pathogenesis by application of chemically synthesized SAG to tobacco leaves. SAG was as active as SA in induction of PR-1 gene expression. This induction was preceded by a transient release of SA, which occurred in the extracellular spaces. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in SAR.     

D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

Traffic COPs and the formation of vesicle coats

Forward and retrograde trafficking of secretory proteins between the endoplasmic reticulum and the Golgi apparatus is driven by two biochemically distinct vesicle coats, COPI and COPII. Assembly of the coats on their target membranes is thought to provide the driving force for membrane deformation and the selective packaging of cargo and targeting molecules into nascent transport vesicles. This review describes our current knowledge on these issues and discusses how the two coats may be differentially targeted and assembled to achieve protein sorting and transport within the early secretory pathway.     

Conformational mimicry: Synthesis and solution conformation of a cyclic somatostatin hexapeptide containing a tetrazole cis amide bond surrogate

Potent, cyclic hexapeptide analogues of somatostatin are generally believed to adopt some common secondary structural features: a II′ β turn at one end of the cycle, and a type VI turn with a cis amide bond at the other. A proposed cis amide surrogate, the 1,5-disubstituted tetrazole, has been placed into a cyclic hexapeptide analog of somatostatin in order to constrain the putative cis amide bond. The final cyclization was done by either chemical or enzymatic means. The product, cyclo(Ala⁶-Tyr⁷-D -Trp⁸-Lys⁹-Val¹⁰-Phe¹¹-Ψ[CN₄]), was found to have 83% of the activity of somatostatin. Solution nmr analysis in DMSO/water revealed that the backbone as well as side chain χ₁ and χ₂ were well ordered. Relaxation matrix methods were used to extract distance restraints from the nuclear Overhauser effect spectroscopy data set, and these were used in a systematic search of torsional space to identify structures consistent with the nmr data. Restrained minimizations of these structures using a number of different force fields produced structures having the expected βII′ turn at D -Trp⁸-Lys⁹ and αβVIa turn in the Phe¹¹-Ψ[CN₄]-Ala⁶ portion of the molecule. The similarity of the minimized structures to those previously reported for cyclic hexapeptide analogues of somatostatin confirms the similarity of the tetrazole geometry to that of the cis amide in solution. © 1995 John Wiley & Sons, Inc.