Synthesis of modified tetrasaccharide sequences of N-glycoproteins

The tetrasaccharides O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D- mannopyranosyl-(1----6)]-O-(4-deoxy-beta-D-lyxo-hexopyranosyl)-(1- ---4)-2- acetamido-2-deoxy-alpha, beta-D-glycopyranose (22) and O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl-(1----6)]-O- beta-D-talopyranosyl-(1----4)-2-acetamido-2-deoxy-alpha, beta-D- glucopyranose (37), closely related to the tetrasaccharide core structure of N-glycoproteins, were synthesized. Starting with 1,6-anhydro-2,3-di-O-isopropylidene-beta-D-mannopyranose, the glycosyl donors 3,6-di-O-acetyl-2-O-benzyl-2,4-dideoxy-alpha-D-lyxo- hexopyranosyl bromide (10) and 3,6-di-O-acetyl-2,4-di-O-benzyl-alpha-D-talopyranosyl bromide (30), were obtained in good yield. Coupling of 10 or 30 with 1,6-anhydro-2-azido-3-O-benzyl-beta-D-glucopyranose to give, respectively, the disaccharides 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2-O-benzyl-4 -deoxy- beta-D-lyxo-hexopyranosyl)-beta-D-glucopyranose and 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2,4-di-O-ben zyl- beta-D-talopyranosyl)-beta-D-glucopyranose was achieved with good selectivity by catalysis with silver silicate. Simultaneous glycosylation of OH-3' and OH-6' of the respective disaccharides with 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl chloride yielded tetrasaccharide derivatives, which were deblocked into the desired tetrasaccharides 22 and 37.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Macrorestriction Analysis of Caenorhabditis Elegans Genomic DNA

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a ``macrorestriction mapping' procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is <220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located ~400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.     

Theoretical temperature distributions produced by an annular phased array-type system in CT-based patient models

Theoretical calculations for the specific absorption rate (SAR) and the resulting temperature distributions produced by an annular phased array (APA)-type system are made. The finite element numerical method is used in the formulation of both the electromagnetic (EM) and thermal boundary value problems. A number of detailed two-dimensional patient models based on CT-scan data from the pelvic, visceral, and thoracic regions are generated to simulate a variety of tumor locations and surrounding normal tissues. The SAR values from the EM solution are put into the bioheat transfer equation, and steady-state temperature distributions are calculated for a wide range of blood flow rates. Based on our theoretical modeling, the APA shows no preferential heating of superficial over deep-seated tumors. However, in most cases for all three regions of the human trunk only fair thermal profiles (therapeutic area near 60%) are obtained in tumors with little or no blood flow and poor temperature patterns (therapeutic area less than 50%) are found in tumors with moderate to high perfusion rates. These theoretical calculations should aid the clinician in the evaluation of the effectiveness of APA-type devices in heating tumors located in the trunk region.     

STRUCTURE OF EXTRACELLULAR POLYSACCHARIDES PRODUCED BY A SOIL CRYPTOMONAS SP. (CRYPTOPHYCEAE)1

The Hindak strain of a Cryptomonas species (Cryptophyceae) produces extracellular polysaccharides. Because there is no information on the structure of these compounds in the Cryptophyceae we conducted structural studies. Gas–liquid chromatographic analyses showed that the polysaccharide is composed of fucose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, and traces of 3-O-methyl galactose. The polysaccharide was separated into two subtractions by ion-exchange chromatography. Fraction A consisted mainly of 1,3-linked galactose units and 1,4-linked galacturonic acid. Unlike fraction B, fraction A did not have xylose, 3-O-methyl galactose, or glucuronic acid. Also, its degree of branching was low compared to that of fraction B. Only traces of sulfate were present infraction A, but fraction B was 10–15% sulfated. Protein was approximately 1% in both fractions. These polysaccharides appear to be a novel type of polymer in algae.     

Dynamics of dissolved organic nitrogen in subalpine Castle Lake,California

Dissolved organic nitrogen (DON) concentrations were measured in meso-oligotrophic Castle Lake, California during the ice-free season in 1982, 1983 and 1984. No consistent relationships were found between DON and particulate-N, primary productivity rates or chlorophyll concentrations. However, increases in DON concentrations were observed in the early growing season of 1982 and 1984 when water temperature was rising and a diatom bloom was senescing. DON increased at a high rate (0.31 mg atom N m^–3 day^–1), and then rapidly disappeared. Sediment released appared to be the most important source of DON. Dissolved free amino acids were always less than 7.5% of the DON pool, and did not vary in the composition of specific amino acids during the growing season.     

The effect of demulsifiers on lysozyme extraction from hen egg white using reverse micelles

The liquid–liquid extraction of protein from buffered aqueous phases using reverse micelles (RM) has been extensively researched from a fundamental point of view. However, very little effort has been expended at scaling up this process for the extraction of real fermentation broth. When real broths are used with reverse micellar phases there are major problems with emulsion formation. In this study the effect of a variety of demulsifiers on lysozyme extraction was evaluated in terms of their influence on the separating properties of the emulsion, water content (W _o ), and, extraction yield and kinetics from both buffer and hen egg white. In addition, the use of a low shear contactor (a Graesser or `raining bucket') was assessed in terms of its suitability as a RM contactor. It was found that most of the demulsifiers reduced the settling time of the emulsion, and enhanced the yield and kinetics of lysozyme extraction from hen egg white. It was hypothesised that this was due to the demulsifier displacing the lysozyme from the interface and preventing the protein unfolding and precipitating. This effect was found to depend on both the generic type of demulsifier, and its concentration.     

Status of genome projects for nonpathogenic bacteria and archaea

Since the first microbial genome was sequenced in 1995, 30 others have been completed and an additional 99 are known to be in progress. Although the early emphasis of microbial genomics was on human pathogens for obvious reasons, a significant number of sequencing projects have focused on nonpathogenic organisms, beginning with the release of the complete genome sequence of the archaeon Methanococcus jannaschii in 1996. The past 18 months have seen the completion of the genomes of several unusual organisms, including Thermotoga maritima, whose genome reveals extensive potential lateral transfer with archaea; Deinococcus radiodurans, the most radiation-resistant microorganism known; and Aeropyrum pernix, the first Crenarchaeota to be completely sequenced. Although the functional characterization of genomic data is still in its initial stages, it is likely that microbial genomics will have a significant impact on environmental, food, and industrial biotechnology as well as on genomic medicine.