Alpha-methyl Mannose Research Articles

Effect of carbohydrates on the adhesion of Bordetella bronchiseptica to the respiratory epithelium in rabbits

This study proposes an ecological approach for preventing respiratory tract infections caused by Bordetella bronchiseptica in mammals using a mixture of carbohydrates. In an in vivo study, 51-day-old New Zealand rabbits were treated with a solution containing 1 × 107 CFUs of B. bronchiseptica and 250 μg of one of the following carbohydrates: N acetylglucosamine (GlcNAc), N acetylgalactosamine (GalNAc), alpha methyl mannose (AmeMan), alpha methyl glucose (AmeGlc) and sialic acid (Neu5AC). Positive (B. bronchiseptica) and negative (Physiological Saline Solution (PSS)) controls were included. Animals treated with GlcNAc or AmeGlc showed no clinical signs of infection and exhibited a significant reduction (p < 0.05) in the severity of microscopic lesions evaluated in the nasal cavity and lung compared with the positive controls. Additionally, the presence of bacteria was not detected through microbiological isolation or PCR in the lungs of animals treated with these sugars. Use of a mixture of GlcNAc and AmeGlc resulted in greater inhibition of microscopic lesions, with a significant reduction (p < 0.05) in the severity of these lesions compared to the results obtained using individual sugars. Furthermore, the bacterium was not detected through microbiological isolation, Polymerase Chain Reaction (PCR) or indirect immunoperoxidase (IIP) in this group.

Quantitative assay for evaluating anti‐clumping reagents

A model system was developed to quantitatively identify reagents that unclump cells (yeast, Saccharomyces cerevisiae). Biofilms, infections and cancer cells in the circulation often involve a clumped or adhering state. Reagents that unclump cells might help lead to the development of anti‐biofilm, anti‐infection and anti‐cancer cell survival drugs. Various sugars and an amino acid (0 mg/ml – 20 mg/ml) were tested for their ability to unclump low pH (2.4), flocculated, formaldehyde (1%) fixed yeast in distilled water. The yeast with and without the reagents were stirred in 0.5 ml droplets on glass microscope slides in distilled water and percentages of single yeast cells (vs clumped yeast) were recorded over a 60 min time course by focusing on the edge of the droplets in 923 total trials. Alpha methyl glucose, alpha methyl mannose and D‐glucose increased the percentage of single cells over controls in the absence of reagents (p less than 0.05), while L‐arginine did not and D‐fucose, alpha lactose and sucrose had inconsistent effects. Most important in this study was the development of an essentially no‐cost, remarkably simple assay, to quantitatively assess reagents that could clump or unclump cells (supported by NSF Presidential Award 0731633, NIH NIGMS SCORE S0648680, RISE, MARC, Joseph Drown Foundation, Sidney Stern Memorial Trust).

Kinetics of yeast dissociation from lectin beads: I. alpha methyl mannose

Here we examine the kinetics of alpha methyl mannose induced dissociation of yeast (Saccharomyces cerevisiae) that have been previously allowed to bind to concanavalin A (Con A) derivatized agarose beads, a model for eluting glycans from Con A beads in purification protocols and for releasing pathogens from human cells. This study tests the statistical reliability of this model using one glycan concentration with hundreds of replicate experiments. Future dose‐response studies will precisely identify the reagents that are most effective at the lowest concentrations. In 963 trials, yeast release from the Con A beads was measured by counting yeast remaining on single yeast bound beads in the presence and absence of 1.5M alpha methyl mannose over a 60 min time course. Two‐ sample t tests showed significant differences in the amount of yeast remaining bound in the sugar versus control (no sugar) samples after 20 min (p less than 0.0001), 40 min (p less than 0.0001) and 60 min (p less than 0.0001), where the sugar caused substantially more dissociation than in its absence. Repeated measures‐ANOVA indicated a significant effect of the glycan treatment on the number of bound yeast (F= 624, p less than 0.0001), as well as a significant interaction between the treatment and time elapsed (F= 339, p less than 0.0001). This is therefore a reliable, simple, precise and inexpensive model for dissociation kinetic studies. Two other studies in these proceeding using different reagents support this conclusion (Supported by NIH NIGMS SCORE (S0648680), RISE, MARC, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).

A novel microdissection model for probing cellular interactions. V. Free Mannan

The adhesion between the NIH model sea urchin embryo roof of the blastocoel and tip of the archenteron is a cellular interaction that has interested investigators for over a century, yet its molecular basis is unknown. Here, 102 blastocoel roofs and archenterons were microdissected out of 102, 54 hr Lytechinus pictus embryos that adhered to each other when placed together. 25 mg per ml free mannan was added to the adhered roofs and archenterons. They remained adhered and then were pulled apart and placed together again in the free mannan solution. All 102 roofs and archenterons adhered once again, the mannan possibly bridging mannan‐binding receptors on both components. As reported in Part III of these studies, mannan conjugated agarose beads also adhered to the dissected roofs and archenterons and here this work was extended to 65, 54 hr L.pictus embryos. Part I of these studies indicated that alpha methyl mannose blocked the cellular interaction. Part II of these studies showed that mannose‐binding lectins could block the interaction. Taken together, this set of studies suggests that mannose‐containing glycans and mannose‐binding receptors may be involved in the cellular interaction between the archenteron and blastocoel roof. This microdissection method, that removes the components of adhesive events from intact embryos, is novel and likely to lead to a better understanding of the molecular basis of cellular interactions (supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology FIRE course funding).

A novel microdissection model for probing cellular interactions. I. alpha methyl mannose blocks the cellular interaction

Adhesion between the archenteron tip and blastocoel roof in the NIH designated sea urchin embryo model is a cellular interaction that has interested investigators for over a century, but its molecular basis is not understood. Here we microdissect the two components of this cellular interaction and use alpha methyl mannose to map molecular groups that may be involved in mediating the cellular interaction. Whole 48–54 hour fixed Lytechinus pictus embryos were washed three times and then dissected in pH 8.0 artificial sea water (ASW), 15°C. The roofs of the blastocoel and the tips of the archenteron were put together and observed to stick together. In over 50 separate trials using bound sets of roof and archenterons attached together, either 0.2 M or 1.0 M alpha methyl mannose was added to the pieces. The pieces remained together. However in 100% of the cases, manually separating the pieces in alpha methyl mannose at either molarity resulted in blocking the rebinding of the blastocoel roofs and archenteron tips.Re‐binding occurred in the absence of the sugar. The results suggest that alpha methyl mannose binding receptors are involved in mediating the cellular interaction. These studies offer a novel approach to map glycans and glycan binding partners that may be functionally important. By microdissecting the components of cellular interactions out of the embryo proper, these components can be probed in pristine media away from factors in intact embryos that could confuse results (Supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding).

A novel microdissection model for probing cellular interactions. III. mapping with Con A and mannan beads

Adhesion between the archenteron tip and blastocoel roof in the NIH designated sea urchin embryo model is a cellular interaction that has interested investigators for over a century, but its molecular basis is not understood. Here we microdissect the two components of this cellular interaction and use concanavalin A (Con A) and mannan derivatized agarose beads to map molecular groups that may be involved in mediating the cellular interaction. In over 50 separate trials, the results were either 100% binding or no binding, and indicated that Con A derivatized and mannan derivatized agarose beads bound to formaldehyde fixed dissected archenterons and roofs in both sterile distilled water (ddH2O) and pH 8.0 artificial sea water (ASW), 15°C, while 0.2 M alpha methyl mannose blocked the binding. Previous work indicated that fixed tissue displayed the same cell surface properties as live tissue. The results suggest that both Con A binding ligands and mannan binding partners are present on the two components of the cellular interaction. These studies offer a novel approach to map glycans and glycan binding partners that may be functionally important. By microdissecting the components of cellular interactions out of the embryo proper, these components can be probed in pristine media away from factors in intact embryos that could confuse results (Supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding).

Human brain endothelial ATP synthase β-subunit is mannose-insensitive binding target of FimH

Binding of meningitis-causing Escherichia coli K1 to human brain microvascular endothelial cells (HBMEC) contributes to traversal of the blood-brain barrier, which occurs in part by the mannose-sensitive binding of FimH. In this study, we showed that FimH also binds to HBMEC, independent of mannose, and identified ATP synthase beta-subunit and actin proteins from the surface biotinylated HBMEC as the mannose-insensitive binding targets for FimH. Co-immunoprecipitation experiments in the presence of alpha-methyl mannose verified the binding of FimH to ATP synthase beta-subunit of HBMEC. ATP synthase beta-subunit antibody decreased E. coli K1 binding to HBMEC in the presence of alpha-methyl mannose. Taken together, these findings demonstrate that FimH of E. coli K1 binds to HBMEC in both mannose-sensitive and -insensitive manner.

Activity of Fixed Lectin Beads, A Drug Testing Model

Lectin derivatized agarose beads are widely used for glycan purification, cell surface analysis, and in the development of anti‐infection and anti‐cancer drugs. These beads have limited shelf life, primarily because lectins, like other proteins, can degrade over time. Here we ask, can fixed lectin beads maintain their binding properties, thereby probably extending their shelf lives? We have already shown that fixed yeast display the same binding properties as live yeast. Concanavalin A (Con A) derivatized agarose beads were fixed with 3.7% formaldehyde in distilled water, washed three times, and their binding to mannose (a Con A binding sugar) rich yeast (Saccharomyces cerevisiae) was compared with unfixed washed beads, in the presence and absence of alpha methyl mannose. In scores of experiments, examining and counting thousands of yeast bound to both bead types, it was found that bead fixation did not inhibit yeast binding, while alpha methyl mannose reduced binding. The results suggest that formaldehyde fixed beads are as active as unfixed beads, offering promise that the shelf life of the beads could be greatly extended. The yeast‐lectin bead system is a useful model for assessing lectin‐glycan interactions, because unlike free glycan‐containing molecules, yeast binding is quickly, precisely and easily assessed by eye, without the need for expensive assay systems. Assays are carried out in 0.2 ml droplets on glass slides, using the flat ends of toothpicks to add beads and yeast and mix samples, plus and minus sugar solutions at varying concentrations. See Zem, et al. (Acta Histochem 108: 311‐317, 2006) for a detailed description of this assay system (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).

Multivalent Carbohydrate Recognition on a Glycodendrimer‐Functionalized Flow‐Through Chip

Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.

Parameters Affecting Cell Binding to Lectin Beads in Carbohydrate‐Based Drug Development

Using a model system of washed yeast (Saccharomyces cerevisiae) that possess mannose‐rich cell surfaces and washed concanavalin A derivatized agarose beads that preferentially bind to glycans containing mannose/glucose residues, we tested 4 concentrations of alpha methyl mannose and the same 4 concentrations of D‐melezitose (0.025 mM – 25 mM) and a 50/50 mixture of these 2 sugars at the same final concentrations, over a 30 minute time course, assaying how much yeast remained bound to the beads at 10 minute intervals with and without sugar at each sugar concentration tested. In 15,379 replicate experiments, averaging about 300 trials for each sugar at each time tested, alpha methyl mannose and D‐melezitose caused dissociation of yeast from the Con A beads in a concentration dependent manner with an almost linear time course with little dissociation occurring immediately and incrementally increased dissociation occurring after 10,20 and 30 minutes. The highest sugar concentration (25 mM) tested here was most effective in causing dissociation and the 50/50 mixture of sugars was less effective than the single sugars in causing dissociation at the lower concentrations (less than 25 mM). The results suggest that careful attention should be paid to sugar concentration and incubation times in experiments using lectin derivatized beads in molecular purification protocols and in development of carbohydrate‐based drugs and diagnostic tests (Supported by NIH NIGMS SCORE (S06‐48680, NIH MBRS RISE, NIH MARC and the Joseph Drown Foundation).

Change in degree of type 1 piliation of Escherichia coli during experimental peritonitis in the mouse.

To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism.

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.

Receptors for pea and lentil lectins on human lymphoid cells: demonstration of distinct lectin-defined cell subclasses.

Lectins are useful probes for studying cell surface glycoconjugates. Pea (PL) and lentil (LL) lectin each requires for binding a fucosyl- and two alpha-mannosyl residues in core regions of glycopeptides, but differences in outer chain carbohydrates may alter their relative binding affinities. We used binding studies with [125I]-PL and LL and flow cytometry with fluorescein-conjugated (FITC)-PL and -LL to study their interactions with peripheral lymphocytes. Binding of both lectins to lymphocytes was saturable, reversible, and inhibited by alpha-methyl mannose. Scatchard analyses were consistent with two classes of receptors for each lectin. Flow cytometric analyses demonstrated that cell to cell receptor densities varied. Sixty-five percent of lymphocytes bound PL (mean 2 X 10(6) receptors/cell) and 45% bound LL (mean 3 X 10(6) receptors/cell). Competition studies demonstrated mutual inhibition, but flow cytometry revealed persistent FITC-PL or -LL binding depsite 20-fold molar excess of the other lectin. Distributions of receptors for PL and LL on lymphocytes were as follows: 45% of lymphocytes bound both PL and LL; 20% of lymphocytes bound PL alone; 35% of lymphocytes bound neither PL nor LL. Despite similar binding requirements for PL and LL and overlap between their receptors on lymphocytes, there appear to be subsets of receptors specific for each lectin. These results may reflect abilities of PL and LL to discriminate subtle carbohydrate differences on lymphocyte surfaces.

Role of sperm-surface glycoproteins in gamete recognition in two mouse species.

In-vitro fertilization and sperm binding to the zona pellucida within and between Mus musculus (laboratory mouse) and Mus caroli (Asian wild field mouse) were examined. Maximum homologous sperm binding in M. musculus was 12.3 +/- 0.3 spermatozoa bound/egg and in M. caroli, 9.2 +/- 0.2. In heterologous crosses, sperm-zona binding was significantly reduced (M. musculus eggs with M. caroli spermatozoa, 1.9 +/- 0.14; M. caroli eggs with M. musculus spermatozoa, 5.5 +/- 0.2). Homologous sperm-zona interaction was inhibited by different sugars in the two mouse species (M. caroli by alpha-methyl mannose, M. musculus by sialic acid). The data suggest that gamete recognition is highly species specific and that hapten sugars play a significant role in sperm-zona binding.

Identification of leukemia-associated inhibitory activity as acidic isoferritins. A regulatory role for acidic isoferritins in the production of granulocytes and macrophages.

Acidic isoferritins have been identified as leukemia-associated inhibitory activity (LIA), which suppresses colony and cluster formation of colony-forming unit-granulocyte macrophages from normal donors but not from patients with leukemia. LIA was detected in all ferritin preparations tested, including ferritin isolated from normal heart, spleen, liver, and placental tissues, and from the spleens of patients with chronic myelogenous leukemia and Hodgkin's disease. Purified preparations of LIA were composed almost entirely of acidic isoferritins, as determined by immunoassay, radioimmunoassay, and isoelectric focusing. The inhibitory activity in the LIA and ferritin samples was inactivated by a battery of antisera specific for ferritin, including those prepared against acidic isoferritins from normal heart and spleen tissues from patients with Hodgkin's disease, and those previously absorbed with basic isoferritins. Antisera absorbed with acidic isoferritins did not inactivate the inhibitory activity. Separation of LIA and chronic myelogenous leukemia and normal spleen ferritin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing confirmed that the regions of peak inhibitory activity corresponded in each to an apparent molecular weight of approximately 550,000 and to a pI value of 4.7. Similar physicochemical characteristics included inactivation by methods that dissociate ferritin molecules into subunits and by treatment with trypsin, chymotrypsin, pronase, and periodate. The purified preparations were extremely stable to heat treatment. The glycoprotein nature of the inhibitory activity was substantiated because it bound to concanavalin A-Sepharose and was eluted off by alpha-methyl mannose. Inhibitory activity of the activity of the acidic isoferritins was detected at concentrations as low as 10(-17)-10(-19) M and iron saturation did not appear to be necessary for its action. These results implicate acidic isoferritins in the regulation of normal myelopoiesis and suggest a role for them in the progression of leukemia.

5'-Nucleotidase activity of mouse peritoneal macrophages. II. Cellular distribution and effects of endocytosis.

The diazonium salt of sulfanilic acid (DASA) can inactivate about 80% of the total 5'-nucleotidase of viable macrophages. The remaining 20% can be inactivated if the cells are first lysed in detergent, and presumably represents an intracellular pool of 5'-nucleotidase. The bulk of this pool may represent cytoplasmic vesicles derived from plasma membrane by endocytosis. This internal compartment is expanded up to threefold immediately after the cells have ingested a large latex load. This is consistent with previous observations on the internalization of 5'-nucleotidase in latex phagosomes. In latex-filled cells this intracellular pool of enzyme is inactivated over a few hours, and the cells then slowly increase their enzyme activity to nearly normal levels. However, 24 h after latex ingestion the metabolism of 5'-nucleotidase in these recovered cells is abnormal, as the rate of enzyme degradation is about twice the normal rate, and the DASA-insensitive enzyme pool in these cells is strikingly diminished. This may reflect effects of the accumulated indigestible particles on the fate of incoming pinocytic vesicles or on newly synthesized plasma membrane precursor. Another endocytic stimulus, concanavalin A, also reduces the total cell 5'-nucleotidase activity. This effect, which is time and temperature dependent, can be prevented by the competitive sugar alpha-methyl mannose. The concanavalin A inhibition can be reversed in the absence of new protein synthesis or in cells cultivated in serum-free conditions. It is not known whether the effect of concanavalin A on 5'-nucleotidase depends upon the interiorizaiton of plasma membrane or is strictly associated with events at the cell surface.