A Rapid High-Resolution High-Performance Liquid Chromatographic Method for Separating Glycan Mixtures and Analyzing Oligosaccharide Profiles

A sensitive and reproducible HPLC technology has been developed, capable of resolving sub-picomolar quantities of mixtures of fluorescently labeled neutral and acidic glycans simultaneously and in their correct molar proportions. The elution positions of standard glycans were determined in glucose units with reference to a dextran ladder, and incremental values for the addition of monosaccharides to oligosaccharide cores were calculated. This information was used to interpret the full oligosaccharide profiles of glycoproteins in a predictive manner based on arm specificity, linkage, and monosaccharide composition. The technique was applied to several systems. For example, a family of glycans isolated from the human parotid gland was extensively resolved on the basis of type and extent of outer arm fucosylation. Second, a serum IgG glycan pool was resolved into 20 peaks which were analyzed simultaneously by sequentially digesting the pool of sugars with exoglycosidase enzymes. In addition, alterations in the glycosylation of IgG associated with rheumatoid arthritis were directly monitored. The reproducibility of the separation system, the predictability of glucose unit values, and the quantitative response of the detection system for individual fluorescently labeled glycans also allowed the automatic analysis of neutral sugars using combinations of enzymes as in the reagent array analysis method (RAAM). In addition, the simultaneous resolution of both acidic (sialylated) and neutral products from the RAAM digestion allowed direct analysis of sialylated glycans, eliminating the previous need to remove sialic acid residues in a preliminary step. Overall, the technologies described here represent a significant advance toward faster, more automated, and more detailed glycan analysis.     

Genetic map of Salmonella typhimurium, edition VIII.

We present edition VIII of the genetic map of Salmonella typhimurium LT2. We list a total of 1,159 genes, 1,080 of which have been located on the circular chromosome and 29 of which are on pSLT, the 90-kb plasmid usually found in LT2 lines. The remaining 50 genes are not yet mapped. The coordinate system used in this edition is neither minutes of transfer time in conjugation crosses nor units representing "phage lengths" of DNA of the transducing phage P22, as used in earlier editions, but centisomes and kilobases based on physical analysis of the lengths of DNA segments between genes. Some of these lengths have been determined by digestion of DNA by rare-cutting endonucleases and separation of fragments by pulsed-field gel electrophoresis. Other lengths have been determined by analysis of DNA sequences in GenBank. We have constructed StySeq1, which incorporates all Salmonella DNA sequence data known to us. StySeq1 comprises over 548 kb of nonredundant chromosomal genomic sequences, representing 11.4% of the chromosome, which is estimated to be just over 4,800 kb in length. Most of these sequences were assigned locations on the chromosome, in some cases by analogy with mapped Escherichia coli sequences.     

N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line

To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.     

The chromosome constitution of 1000 human spermatozoa

Summary Chromosomal analysis of 1000 spermatozoa from 33 normal men was performed using in vitro fertilization of zonafree golden hamster eggs. The frequency of abnormal sperm complements was 8.5%: 5.2% were aneuploid and 3.3% had a structural chromosome abnormality. The frequencies of hyperhaploid (2.4%) and hypohaploid (2.7%) sperm complements were not significantly different and all chromosome groups were represented among the aneuploid complements. The majority (22/33) of structurally abnormal complements had a chromosome break. The percentages of X and Y-bearing sperm were 53.9% and 46.1%, which is significantly different from the expected one to one ratio.     

The role of the 2H4 molecule in the generation of suppressor function in Con A-activated T cells

The molecular basis for the suppression generated in a concanavalin A (Con A)-activated T cell culture remains unknown. In this study, we have attempted to determine whether the 2H4 and 4B4 molecules on Con A-activated T cells play some role in the generation of suppression by such cells. We have shown that Con A-activated suppressor cells belong to the 2H4+ subset of T cells but not the 4B4+ (2H4-) subset. Con A-activated T cells exerted their optimal suppressor function on day 2 in culture, a time at which the expression of 2H4 on such cells was maximal and 4B4 was minimal. Furthermore, the stimulation of T cells with the higher concentration of Con A generated the stronger suppressor function. At the same time, both 2H4 expression and density were increased and 4B4 expression and density were decreased on such Con A-activated T cells. More importantly, the treatment of Con A-activated T cells with anti-2H4 antibody but not with anti-4B4, anti-TQ1, or anti-T4 antibodies can block the suppressor function of such cells. Taken together, the above results strongly suggest that the 2H4 molecule itself may be involved in the generation of suppressor function in Con A-activated T cells. The 2H4 antigen on such cells was shown to be comprised of 220,000 and 200,000 m.w. glycoproteins. Thus this study indicates that the 220,000 and 200,000 m.w. structure of the 2H4 molecule may itself play a crucial role in the generation of suppressor signals of Con A-activated cells.     

Measurement of Mercury Methylation in Lake Water and Sediment Samples

The production of various eremophilane-type sesquiterpenes by Penicillium roqueforti strains has allowed us to propose a biochemical pathway for PR toxin synthesis. A time-course study of P. roqueforti metabolite production by high-performance liquid chromatography was performed to check this hypothetical pathway. The results obtained suggested that eremofortin C was the direct precursor of PR toxin in the P. roqueforti cell. Attempts to determine the amount of PR toxin in the mycelium failed. It was shown that the absence of PR toxin in mycelium was due to its instability during the extraction procedure.     

Two new species of Paramachaerium (Leguminosae) and a brief résumé of the genus

Paramachaerium Ducke, a genus of neotropical trees, has been known hitherto from three species,P. schomburgkii (Guyana and Brazil),P. ormosioides (French Guiana and Brazil), andP. gruberi (Panamá). Two new species are herein described,P. krukovii (western Brazil) andP. schunkei (Peru).