Carbohydrate Markers Research Articles

Ganglioside SSEA-4 in Ewing sarcoma marks a tumor cell population with aggressive features and is a potential cell-surface immune target

Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.

Convergent synthesis of functionalized derivatives of stage-specific embryonic antigens 3 & 4

Stage-specific embryonic antigens (SSEAs) are carbohydrate markers that have diverse roles in embryonic development. However, the exact roles of SSEAs remain unclear. To obtain mechanistic insights into their roles, we aimed to develop functionalized SSEA glycan analogs via chemical synthesis. Herein, we report a convergent synthetic approach for SSEA-3 and SSEA-4 analogs using readily available versatile building blocks. A key step, namely the stereoselective glycosylation of a common tetrasaccharide acceptor, was successfully achieved using a 4-O-Bn Gal donor for SSEA-3 and a Neu-Gal donor for SSEA-4, which were previously developed by our group. The obtained SSEA-3 and SSEA-4 glycans were further functionalized with biotin and deuterated lipid for applications in biological studies. Thus, the findings of this study will facilitate further research on SSEAs.

1411-P: Dysfunction of Adipose Tissue and Testosterone Deficiency in Men with Type 2 Diabetes Mellitus

The prevalence of hypogonadism in men with type 2 diabetes mellitus (T2DM) is higher than in population. Though, the function of adipose tissue in men with hypogonadism and T2DM is still unclear. Aim: To study the function of adipose tissue in men with T2DM and functional hypogonadism. Materials and Methods: We examined 272 men with T2DM (mean age 53.8±2.7 years). Randomization: 1 group - 148 men with hypogonadism, according to the European Association of Urology criteria 2015; 2 group - 124 patients, not having hypogonadism. The groups were comparable by age and treatment of diabetes. Patients underwent clinical examination, analysis of carbohydrate metabolism and markers of adipose tissue function, such as resistin, leptin, adiponectin. Statistical analysis was carried out using the Mann Whitney U-test. Results: The analysis demonstrated that hypogonadal patients had a significantly higher body mass index, waist and hip circumferences. Levels of fasting glucose and HbA1c weren’t statistically different in two groups. At the same time, patients with hypogonadism had more pronounced hyperinsulinemia (16,2 [10,5; 30,2] vs. 12,7 [8,4; 21] μU/ml, p=0,002) and insulin resistance, calculated as HOMA-IR (6,2 [3,8; 11,0] vs. 4,6 [2,8; 7,0], p<0,001), compared to eugonadal men. The analysis revealed an increase in leptin (10,8 [7,2; 17,5] vs. 9,2 [5,8; 14,9] ng/ml, p=0,03) and resistin levels (7,5 [2,9; 13,3] vs. 3,8 [1,8; 6,5] ng/ml, p<0,001) in the 1st group compared to the 2nd. Contrary, levels of adiponectin (2,8 [0,5; 5,7] vs. 4,7 [0,8; 7,9] μg/ml, p=0.006) were higher in group 2 compared to the 1st one. Thus, despite the equal fasting glucose and HbA1c levels, men with T deficiency have more pronounced adipose tissue dysfunction compared to eugonadal men having T2DM. Conclusion: T deficiency leads to adipose tissue dysfunction in men with T2DM. Disclosure I.A. Khripun: None. S.V. Vorobyev: None. E.V. Bova: None. Funding Russian Science Foundation (14-25-00052)

Changes in clinical and biochemical characteristics of polycystic ovary syndrome with advancing age.

ObjectiveTo verify whether aging can modify the clinical and biochemical characteristics of women with polycystic ovary syndrome (PCOS).Material and methodsThis observational cross-sectional study was conducted at the reproductive endocrinology clinics of Julio Muller University Hospital and Tropical Institute of Reproductive Medicine in Cuiabá, MT, Brazil, between 2003 and 2017. Both, 796 PCOS and 444 non-PCOS normal cycling women underwent the same examination. PCOS was diagnosed using the Rotterdam criteria as recommended for adolescent and adult subjects. Anthropometric, metabolic, and endocrinological modifications with aging were initially examined in the two groups: control and PCOS. Further analyses were performed after a 5-year age stratification of data throughout the reproductive period. All participants signed a consent form approved by the local ethical committee.ResultsBiomarkers of adiposity were more remarkable in African descendant PCOS women. Body weight, waist/hip ratio, fat mass, and BMI were higher in PCOS women and tended to increase at all 5 age-strata, between ≤19 and 35 years of age. Serum androgen levels decreased with aging, markedly in PCOS subjects (P < 0.01 for all age-strata comparisons), but remained elevated when compared with the levels found in controls. Carbohydrate markers, triglycerides, and total cholesterol tended to increase over time in PCOS (P < 0.01 for all age-strata comparisons). Total cholesterol also tended to increase with age in non-PCOS women (P = 0.041).ConclusionThe present study has shown that the advancing age influences many features of PCOS women. Biochemical hyperandrogenism, the core criterion recommended in the current systems to define the syndrome, showed statistically significant tendencies to decrease with aging progression but did not normalize. The use of age-adjusted features for the diagnosis of PCOS are recommended.

Abstract LB-178: Oxidation markers in relation to prostate cancer and BPH and progression of prostate cancer

Abstract Prostate cancer (PCa) is the leading cancer affecting men of all races in the U.S. Benign prostatic hyperplasia (BPH) is prostate enlargement and is not malignant. BPH and PCa have similar symptoms and both have elevated PSA levels. A biomarker which can distinguish PCa from BPH will help the diagnosis of PCa and avoid unnecessary biopsies. Further, many PCa patients after prostatectomy can have recurrence. Currently, PSA is the only monitoring marker for recurrence; however, the underlying mechanism for recurrence is unknown. A mechanistic marker that can predict the progression and recurrence this will allow us to design prevention strategies. Many animal studies have linked oxidative stress with PCa progression and recurrence. The aim of this study is to compare the levels of several oxidation markers between PCa and BPH, and compare the levels of oxidation marker in different stages of PCa and determine their associations with PSA velocities after prostatectomy. We have measured multiple oxidation markers including lipid, protein, DNA, carbohydrate and global oxidation markers. We have collected blood samples among 15 BPH and 22 prostate cancer patients who have prostatectomy. We have found that levels of advanced glycation end products (AGEs), a marker of glucose oxidation, formed under nonenzymatic glycation of lipids and proteins, are higher in PCa patients than BPH. However, among PCa patients, AGEs levels are lower among those with Gleason score higher than 7 than those with Gleason score less than 7. Finally, we have found F2-isoprostanes, a well-established lipid oxidation marker, are associated with PSA velocities among PCa patients who had prostatectomy. Our results suggest that AGEs can help distinguish BPH and PCa but are associated with lower grade PCa. Measurement of F2-isoprostanes, a marker of lipid oxidation can predict PCa progression among men with prostatectomy. Citation Format: Tianying Wu, Bruce Bracken, Susan Kasper. Oxidation markers in relation to prostate cancer and BPH and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-178.

Interaction of Mycobacterium tuberculosis H37Rv with Microfold Cell leads to a New Era of infection in Host

Tuberculosis (TB) is widely distributed dangerous disease that spreads at faster rate and caused by Mycobacterium tuberculosis H37Rv (M. tuberculosis H37Rv) which is highly successful lipid and GC rich bacteria. The bacterium gains its success as it utilizes host macrophages for its survival and replication. M. tuberculosis H37Rv uses at least two separate pathways to recruit macrophages. It uses its PGL surface lipid to recruit macrophages through host CCL2 and Micro fold cells to enter deep in tissues. M-cells express many different carbohydrate markers on their surface which helps in cell and pathogen or antigen interaction. These cells transfer substances from gut across epithelium and to immune cells. M-cells have the potential to intricate the life cycle of this pathogen by internalizing pathogen. M-cells are targeted for vaccine to induce immunity and it has experimented on mice, humans and primates. Nanoparticles and microspheres can also be used successfully for drug or vaccine delivery through micro fold cells because microspheres used for vaccine delivery system to increase mucosal antibody responses that provide prolonged therapeutic effects and nanoparticles are easily accepted by cells so these particles also useful in drug delivery. In this manuscript we have describe the significance of M- cells that could be helpful in TB treatment.

Embryoglycan: a highly branched poly-N-acetyllactosamine in pluripotent stem cells and early embryonic cells.

Embryonal carcinoma cells, stem cells of teratocarcinomas, are pluripotent stem cells and also prototypes of embryonic stem cells. Embryonal carcinoma cells contain large amounts of a highly branched poly-N-acetyllactosamine called embryoglycan, which has a molecular weight of approximately 10,000 or greater, and is asparagine-linked. This glycan was found by analyses of fucose-labeled glycopeptides, and its characteristics were established by biochemical analyses. The content of embryoglycan progressively decreases during the in vitro differentiation of embryonal carcinoma cells. Embryoglycan is also abundant in mouse embryonic stem cells and preimplantation mouse embryos, and decreases during embryogenesis. Embryoglycan carries a number of carbohydrate markers of murine pluripotent stem cells. Lewis x markers, such as SSEA-1, 4C9 antigen, and binding sites for Lotus tetragonolobus agglutinin are of particular importance. 4C9 antigenicity requires clustering of Lewis x, best accomplished by poly-N-acetyllactosamine branching, whereas SSEA-1 does not. Although in vivo evidence is lacking, these epitopes have been suggested to participate in cell-to-cell and cell-to-substratum adhesion. Other markers on embryoglycan include α-galactosyl antigens such as ECMA-2, and binding sites for Dolichos biflorus agglutinin, the epitope of which is considered to be identical to Sda antigen, namely, GalNAcβ1-4(NeuAcα2-3)Galβ1-4GlcNAc. While embryoglycan is also present in human teratocarcinoma cells, the carbohydrate markers characterized in human pluripotent stem cells to date are largely carried by glycolipids and keratan sulfate. Information on embryoglycan and markers carried by it may assist in the development of new markers of human pluripotent stem cells and their progenies.

Composition of the organic matter in soils from six pedoclimatic zones of Magdalena (Colombia)

The soils in the NE of the Magdalena province (Colombia) are subject to intense degradation primarily because of the low accumulation rate of soil organic matter (SOM) and its fast turnover. Numerous traditional studies have aimed to mitigate this situation, focusing on the analysis of total SOM contents. The present study aimed to determine SOM quality from six areas under humid and dry tropical forest with different pedoclimatic conditions, using molecular characterization. For each area, sites with different current land use were studied, including forest and cultivated sites. We applied analytical pyrolysis (pyrolysis-GC/MS) to aqueous NaOH-extractable SOM from surface soil samples. One-hundred and ten pyrolysis products were identified and quantified, from the following chemical groups: N-containing compounds (34.1 ± 5.1%), carbohydrate markers (21.1% ± 7.1%), monocyclic aromatic compounds (20.0% ± 4.5%), aliphatic compounds (7.7% ± 1.4%), phenols (7.0% ± 5.0%), polycyclic aromatic compounds (3.6% ± 0.9 %), lignin products (0.3% ± 0.3%) and other compounds (0.2% ± 0.1%). SOM of all studied areas showed a large proportion of N-containing products, indicating a high contribution from microbial material to the SOM. Multivariate statistics allowed for the differentiation between microbial-derived and plant-derived SOM (Factor 1) and the relative proportion of burning residues (Factor 2), and indicated that pedoclimatic conditions exert more influence on SOM composition (cool and humid zones were enriched in microbial SOM while dry zones contained more plant-derived SOM) than land use for the studied soils.

Efficacy and safety of medicinal plants used in the management of diabetes mellitus.

Efficacy and safety of medicinal plants used in the management of diabetes mellitus.

Recent Progress in Carbohydrate Biosynthesis and Function in Relation to Tumor Biology

Recent development in carbohydrate markers and functions are described. Identification of carbohydrate epitope for cancer-specific antibody is introduced. This novel approach involves the key glycosyltransferases that synthesize tumor-associated carbohydrate-antigens and elucidate the biosynthetic pathways. This is the true determination of carbohydrate ligands and glycan array is secondary to determine the epitope. Tumor suppressor activity of carbohydrate is described. Cell surface carbohydrate, which expressed in normal cells is diminished on cancer cells, function as a tumor suppressor. Glycans attached to α-dystroglycan function as laminin-binding glycans. In cancer cells, oncogene downregulates laminin-binding glycans and they do not bind to laminin in extracellular matrix, making cells to mobile. Thus, laminin-binding glycans function to suppress the cell mobility, thereby suppressing tumor formation in normal cells. This article summarizes the recent progress in the regulation of carbohydrate function in cancer cells. Since the review is short and not comprehensive, other several important topics may be missing.

Carbohydrate markers of pancreatic cancer

Pancreatic cancer is the fourth most common cause of death from cancer in the world and the sixth in Europe. Pancreatic cancer is more frequent in males than females. Worldwide, following diagnosis of pancreatic cancer, <2% of patients survive for 5 years, 8% survive for 2 years and <50% survive for only approx. 3 months. The biggest risk factor in pancreatic cancer is age, with a peak of morbidity at 65 years. Difficulty in the diagnosis of pancreatic cancer causes a delay in its detection. It is one of the most difficult cancers to diagnose and therefore to treat successfully. Additional detection of carbohydrate markers may offer a better diagnosis of pancreatic cancer. Carbohydrate markers of cancer may be produced by the cancer itself or by the body in response to cancer, whose presence in body fluids suggests the presence and growth of the cancer. The most widely used, and best-recognized, carbohydrate marker of pancreatic cancer is CA 19-9 [CA (carbohydrate antigen) 19-9]. However, the relatively non-specific nature of CA 19-9 limits its routine use in the early diagnosis of pancreatic cancer, but it may be useful in monitoring treatment of pancreatic cancer (e.g. the effectiveness of chemotherapy), as a complement to other diagnostic methods. Some other carbohydrate markers of pancreatic cancer may be considered, such as CEA (carcinoembryonic antigen), CA 50 and CA 242, and the mucins MUC1, MUC2 and MUC5AC, but enzymes involved in the processing of glycoconjugates could also be involved. Our preliminary research shows that the activity of lysosomal exoglycosidases, including HEX (N-acetyl-β-D-hexosaminidase), GAL (β-D-galactosidase), FUC (α-L-fucosidase) and MAN (α-D-mannosidase), in serum and urine may be used in the diagnosis of pancreatic cancer.

Can pyrolysis-GC/MS be used to estimate the degree of thermal alteration of black carbon?

Very little is known about the macromolecular properties of biomass combustion residues referred to as black carbon (BC). Pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) was performed on: (i) peat from Spain at 400–1200 °C to investigate the effect of charring on pyrolysis fingerprint and (ii) natural charcoal from Laos in order to link molecular information to published chemical and reactivity parameters. Confirming earlier Py-GC/MS studies, the BC in the artificially charred peat and the natural charcoal produced predominantly benzene, toluene, C 2-benzenes, PAHs and benzonitriles. Furthermore, some charcoal samples produced significant amounts of phenols, methoxyphenols, carbohydrate markers, n-alkanes and n-alkenes upon pyrolysis, reflecting non-charred and weakly charred biomass. A series of pyrolysis product ratios related to the degree of dealkylation of the pyrolysis products (benzene/toluene, naphthalene/C 1-naphthalenes, C 1-naphthalenes/C 2-naphthalenes, benzofuran/C 1-benzofurans and benzonitrile/C 1-benzonitrile) increased with increasing artificial charring (peat) and, for the natural charcoal, these ratios were in accordance with established chemical and reactivity parameters related to charring intensity from other methods: proportion of aromatic C obtained from solid state 13C nuclear magnetic resonance spectroscopy (NMR), the proportion of charred material as estimated from NMR in conjunction with a molecular mixing model (NMR–MMM) and the resistance to acid dichromate oxidation. The alkyl side chains of aromatic pyrolysis products are probably inherited from short chain aliphatic C chains that cross link the predominantly aromatic building blocks of BC, and these linkages seem to disappear with increasing charring intensity. Thus, the degree of thermal alteration of BC can be discerned from the pyrolysis fragmentation pattern.

Cell surface sialylation and fucosylation are regulated by the cell recognition molecule L1 via PLCγ and cooperate to modulate embryonic stem cell survival and proliferation

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown. Using a panel of carbohydrate markers, we have shown that cell surface sialylation and fucosylation are upregulated in L1-transfected embryonic stem cells (L1-ESCs). Consistently, the mRNA levels of sialyltransferase ST6Gal1 and ST3Gal4, and fucosyltransferase FUT9 were significantly increased in L1-transfected ESCs. Activation of L1 signaling promoted cell survival and inhibited cell proliferation. ShRNAs knocking down FUT9, ST6Gal1 and ST3Gal4 blocked these effects. A phospholipase Cγ (PLCγ) inhibitor and shRNA reduced ST6Gal1, ST3Gal4 and FUT9 mRNA levels in the L1-ESCs. Thus, embryonic stem cell surface sialylation and fucosylation are regulated via PLCγ by L1, with which they cooperate to modulate cell survival and proliferation.

Structural insights into the innate immune recognition specificities of L- and H-ficolins

Innate immunity relies critically upon the ability of a few pattern recognition molecules to sense molecular markers on pathogens, but little is known about these interactions at the atomic level. Human L- and H-ficolins are soluble oligomeric defence proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. The X-ray structures of their trimeric recognition domains, alone and in complex with various ligands, have been solved to resolutions up to 1.95 and 1.7 A, respectively. Both domains have three-lobed structures with clefts separating the distal parts of the protomers. Ca(2+) ions are found at sites homologous to those described for tachylectin 5A (TL5A), an invertebrate lectin. Outer binding sites (S1) homologous to the GlcNAc-binding pocket of TL5A are present in the ficolins but show different structures and specificities. In L-ficolin, three additional binding sites (S2-S4) surround the cleft. Together, they define an unpredicted continuous recognition surface able to sense various acetylated and neutral carbohydrate markers in the context of extended polysaccharides such as 1,3-beta-D-glucan, as found on microbial or apoptotic surfaces.

Paper pulp delignification using laccase and natural mediators

Three plant phenols, namely acetosyringone, syringaldehyde and p-coumaric acid, were selected as laccase redox mediators to investigate the enzymatic delignification of paper pulp (obtained from kraft cooking of eucalypt wood) in combination with peroxide bleaching. The effects of these natural mediators were compared with those obtained using the synthetic mediator 1-hydroxybenzotriazole. p-Coumaric acid only caused minor increase of pulp brightness and did not lower its kappa number (a rough estimation of the lignin content), whereas, the use of acetosyringone or syringaldehyde as laccase mediators enabled over 15% increase of final brightness and a similar decrease of final kappa number. Pulp delignification by laccase in the presence of the two latter natural mediators was demonstrated by analytical pyrolysis, which does not suffer from interferences by other pulp constituents as kappa number does, showing a preferential removal of lignin marker compounds compared with carbohydrate markers (up to 25% decrease of the corresponding ratio). This technique also revealed a modification of the residual lignin composition in terms of phenylpropane units after the laccase-mediator treatment. The use of laccase in combination with natural mediators, widely available from plant materials and pulping liquors, represents a promising alternative for environmentally friendly delignification of paper pulp.

Carbohydrate antigens expressed on stem cells and early embryonic cells.

Lewis X antigen (Le(X)) is a marker of embryonic stem cells, embryonal carcinoma cells and multipotential cells of early embryos in the mouse. Le(X) is carried by branched, high-molecular weight poly-N-acetyllactosamines (embryoglycan). While embryoglycan is present in human embryonal carcinoma cells, Le(X) is not expressed in human embryonic stem cells, embryonal carcinoma cells or inner cell mass cells. Instead, these cells express SSEA-3 and SSEA-4, both of which are carried by globo-series glycolipids. Le(X) is a marker of primordial germ cells or multipotential stem cells derived from primordial germ cells both in the mouse and human. In other species of vertebrates, Le(X) is widely expressed in early embryonic cells and primordial germ cells, but the mode of expression is not completely conserved among species. Le(X) is expressed in neural stem cells from both humans and mice. Hematopoietic stem cells are not reported to express the above carbohydrate markers. A marker of these cells is CD34, a membrane-bound sialomucin. Another sialomucin, CD164 (MGC-24v) is expressed in hemotopoietic progenitor cells. As a function of Le(X) in stem cells, the promotion of integrin action is proposed, based on analyses of glycoproteins with the marker, cDNA transfection experiments and the inhibitory effects of an anti-Le(X) antibody. Most probably, Le(X) antigen as well as poly-N-acetyllactosamines play roles in the interactions on the same membrane. On the other hand, O-linked oligosaccharides on CD34 and CD164 are probably involved in the regulation of cell adhesion and proliferation via intercellular recognition.

Application of pyrolysis–GC/MS for the characterisation of suspended particulate organic matter in the Mediterranean Sea: a comparison with the Black Sea

Suspended particulate organic matter (SPOM) both from the oligotrophic Mediterranean and from the more productive Black Sea has been analysed by pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) and by such conventional techniques as elemental analysis and fluorometry. The data achieved by the two approaches generally confirmed and complemented each other. The relative concentrations of pyrolysis products (termed markers) characteristic of chlorophyll (CHL), lipid, carbohydrate (CBH), and protein (PROT) components of SPOM have been determined. The vertical distribution of lipid markers was more uniform in the Mediterranean surface waters. Their relative abundance increased rapidly in the oxycline of the Black Sea, reaching their highest levels in the suboxic zone, where the protein composition of the SPOM changed significantly. In both seas, the relative concentrations of CHL markers increased consistently within the CHL maximum zone where the CBH markers were less abundant. No lignin markers from terrestrial vegetation were detected at significant levels in the pyrograms.

Protein-bound carbohydrates on cell-surface as targets of recognition: an odyssey in understanding them.

Multidisciplinary approaches by a number of investigators have established that cell-surface carbohydrates are integral components of recognition systems regulating survival, migration, adhesion, growth and differentiation of various cells. Our own experience and contributions to this exciting field are described. We discovered Endo D as the first endoglycosidase acting on glycoproteins, found complementary specificity of two endoglycosidases (Endo D and Endo H), and applied these enzymes for glycoprotein research. Endo-beta-galactosidase C, which hydrolyzes Galalpha1-3Galbeta1-4GlcNAc xenoantigenic determinant, was later found and molecularly cloned. We also found highly branched poly-N-acetyllactosamines in early embryonic cells, and demonstrated developmentally regulated carbohydrate changes during early mammalian development. The binding site for Dolichos biflorus agglutinin was introduced as a new differentiation marker. Basigin and embigin, two related members of the immunoglobulin superfamily, a sialomucin MGC-24 and other glycoproteins were discovered as carriers of developmentally regulated carbohydrate markers. We proposed enhancement of integrin action as a function of sugar chains with Lewis X epitope, and observed a relationship between the expression of carbohydrate markers and invasive properties of human carcinoma. Midkine, a heparin-binding growth factor, was discovered more recently and its interaction with heparin and oversulfated chondroitin sulfate was elucidated. N-Acetylglucosamine-6-sulfotransferase was cloned and used to reconstitute L-selectin ligands. Gene knockout was applied to reveal in vivo function of basigin, syndecan-4 and chondroitin 6-sulfate. Throughout my research on all these subjects, I have been fortunate in obtaining unexpected observations and enjoying fruitful collaborations.

Mannose-Specific Recognition Mediates Two Aspects of Synaptic Growth of Leech Sensory Afferents: Collateral Branching and Proliferation of Synaptic Vesicle Clusters

The developmental role of carbohydrate markers in the genesis of neuronal networks was studied using leech sensory afferents as a model. Leech sensory afferents express a mannose-containing epitope on their cell surface that is recognized by monoclonal antibody Lan3-2. Previously, the elaboration of sensory arbors in the synaptic neuropil of CNS ganglia was experimentally shown to depend on this mannose marker. Sensory arbors were abolished by perturbing sensory afferents in the intact nervous system with Lan3-2 Fab fragments, a glycosidase, or mannose–BSA. To understand the cytological mechanisms underlying mannose-specific recognition for synaptogenesis, we have now studied the effects of antibody perturbation at the ultrastructural level in the sensory afferent target region. A characteristic signature of a normal sensory afferent is its profuse collateral branching, which, with ongoing development, is replaced by a single widened process, the sensory trunk, which possesses numerous synaptic vesicle clusters. The inhibition of mannose-specific recognition leads to a rapid, major reorganization of different stages of sensory afferent growth. Collateral branches at the distal growing region are reduced three- to fourfold. The pruned axons grow at an accelerated rate. Developmentally older sensory trunks experience a threefold reduction in synaptic vesicle clusters. These responses suggest that depriving sensory afferents of mannose-specific recognition aborts their synaptogenesis and causes them to resume behavior typical of tracking through axonal tracts. The current findings also suggest that the mannose marker, by promoting both collateral branching andthe proliferation of synaptic vesicle clusters, plays a critical role in two stages of sensory afferent synaptogenesis.

Genetic approaches to the detection of contaminants in Escherichia coli fermentations.

In facilities which cultivate more than one rDNA organism, contamination by the same species is difficult to detect. Since the same species may be producing a different product in an adjacent fermentor or in the same vessel in a subsequent procedure, the possibility of cross-product contamination must be considered. Here we describe a simple, sensitive, and reliable technique for the detection of same-species contamination. The assay uses negative genetic markers such as the inability to use a carbohydrate, e.g., ribose. When the facility is managed to use the ribose marker for only one product, this culture can be plated on ribose minimal medium to allow rapid and sensitive detection of contaminants. If the facility is used for several rDNA products, multiple carbohydrate markers per strain can be used so that a limited number of markers can differentiate among larger host collections. The approach was developed and tested using Escherichia coli as the host organism. If hosts with auxotrophies, e.g., for amino acids, are used in the facility, the plate medium can be supplemented. When this technique is combined with existing methods for detecting different species and bacteriophage contamination, all three classes of biological contamination can be detected.