A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria.

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3-4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A-(GlcNAc)2 complex in a 'half-open' conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane-transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.

Taste responsiveness of Western chimpanzees (Pan troglodytes verus) to five food-associated saccharides.

Using a two-bottle choice test of short duration, we determined taste preference thresholds for sucrose, fructose, glucose, lactose, and maltose in three Western chimpanzees (Pan troglodytes verus). Further, we assessed relative preferences for these five saccharides when presented at equimolar concentrations and determined taste preference difference thresholds for sucrose, that is, the smallest concentration difference at which the chimpanzees display a preference for one of the two options. We found that the chimpanzees significantly preferred concentrations as low as 20 mM sucrose, 40 mM fructose, and 80 mM glucose, lactose, and maltose over tap water. When given a choice between all binary combinations of these five saccharides presented at equimolar concentrations of 100, 200, and 400 mM, respectively, the animals displayed significant preferences for individual saccharides in the following order: sucrose > fructose > glucose = maltose = lactose. The taste difference threshold for sucrose, expressed as Weber ratio (ΔI/I), was 0.3 and 0.4, respectively, at reference concentrations of 100 and 200 mM. The taste sensitivity of the chimpanzees to the five saccharides falls into the same range found in other primate species. Remarkably, their taste preference thresholds are similar, and with two saccharides even identical, to human taste detection thresholds. The pattern of relative taste preferences displayed by the chimpanzees was similar to that found in platyrrhine primates and to the pattern of relative sweetness as reported by humans. Taken together, the results of the present study are in line with the notion that taste sensitivity for food-associated carbohydrates may correlate positively with phylogenetic relatedness. Further, they support the notion that relative preferences for food-associated carbohydrates, but not taste difference thresholds, may correlate with dietary specialization in primates.

Age-Related Dry Eye Lactoferrin and Lactobionic Acid.

Dry eye is the most prominent pathology among those involving the ocular surface: a decrease of the aqueous (less frequent) or the lipid (more frequent) component of the tear film is the cause of the diminished stability of tears that is observed in this pathology. Dry eye shows a clear distribution linked to both sex (being more frequent among women) and age (increasing with aging). Therefore, specific treatments taking into account the etiology of the disease would be desired. The role of lactoferrin and its functional mimetic lactobionic acid are reported here as a possible remedy for age-related dry eye.

Potassium, not lepidimoide, is the principal 'allelochemical' of cress-seed exudate that promotes amaranth hypocotyl elongation.

Background and Aims: Imbibed cress ( Lepidium sativum L.) seeds exude 'allelochemicals' that promote excessive hypocotyl elongation and inhibit root growth in neighbouring competitors, e.g. amaranth ( Amaranthus caudatus L.) seedlings. The major hypocotyl promoter has recently been shown not to be the previously suggested acidic disaccharide, lepidimoic acid (LMA), a fragment of the pectic polysaccharide domain rhamnogalacturonan-I. The nature of the hypocotyl promoter has now been re-assessed. Methods: Low-molecular weight cress-seed exudate (LCSE) was fractionated by high-voltage electrophoresis, and components with different charge:mass ratios were tested for effects on dark-grown amaranth seedlings. Further samples of LCSE were size-fractionated by gel permeation chromatography, and active fractions were analysed electrophoretically. Key Results: The LCSE strongly promoted amaranth hypocotyl elongation. The active principle was hydrophilic and, unlike LMA, stable to hot acid. After electrophoresis at pH 6·5, the only fractions that strongly promoted hypocotyl elongation were those with a very high positive charge:mass ratio, migrating towards the cathode 3-4 times faster than glucosamine. Among numerous naturally occurring cations tested, the only one with such a high mobility was potassium. K + was present in LCSE at approx. 4 m m , and pure KCl (1-10 m m ) strongly promoted amaranth hypocotyl elongation. No other cation tested (including Na + , spermidine and putrescine) had this effect. The peak of bioactivity from a gel permeation chromatography column exactly coincided with the peak of K + . Conclusions: The major 'allelopathic' substance present in cress-seed exudate that stimulates hypocotyl elongation in neighbouring seedlings is the inorganic cation, K + , not the oligosaccharin LMA.

Sugar and amino acid preference in the black garden ant Lasius niger (L.).

The mutualistic relationship that the garden ant Lasius niger (L.) establishes with trophobiotic homopterans makes this ant an unwelcome host in commercial crops, as ants improve the survival of homopteran pests from which they collect honeydew as a source of carbohydrates. Because the offering of alternative sugar sources can be used to disrupt this relationship, the present study explored L. niger's preference towards sugar and amino acid components that may be used in sugar solutions to increase their attractiveness. We tested the ant's preference between basic sugars (mono- and disaccharides) used as main ingredients and attractants (trisaccharides and amino acid (AA) sources) added to basic sugar in small amounts. Results showed that ants preferred disaccharides over monosaccharides, and that trisaccharides increased the attractiveness of sucrose solutions, albeit not when a protein source was added to the mix. In the case of AA sources, ants preferred components with a more diverse composition. In conclusion, trisaccharides and AA sources can be used to increase the attractiveness of sugar solutions, leading to the development of solutions that when supplied in artificial feeders can out-compete honeydew and disrupt harmful ant-homopteran mutualisms in agriculture.

Characteristics of glycosaminoglycans in chicken eggshells and the influence of disaccharide composition on eggshell properties.

Glycosaminoglycans (GAG) are linear, highly negatively charged polysaccharides that may perform an important role in biomineralization. GAG were isolated from chicken eggshell membranes and calcified shells. Disaccharide compositional analysis was performed using liquid chromatography-mass spectrometry. All 4 groups of GAG - hyaluronan (HA), keratan sulfate (KS), chondroitin sulfate (CS), and heparan sulfate (HS) - were detected in shell membranes and in calcified shells. HA was the most plentiful GAG in shell membranes, and CS was the most abundant in calcified shells. The CS present, in both membranes and calcified shells, consisted primarily of 6SCS-C, 4SCS-A, and 0SCS-0 disaccharides. Neither 4S6SCS-E nor 2SCS was detectable in shell components. Small amounts of 2S4SCS-B were detected in membranes and TriSCS, and 2S4SCS-B and 2S6SCS-D were detected in calcified shells. HS in calcified shells contained all disaccharides except for 2S6S. In shell membranes, HS contained primarily NS and 0S as well as small amounts of TriS, NS2S, NS6SHS, and 6S, but neither 2S6S nor 2S was detectable. The disaccharide composition of membrane CS, as well as membrane and calcified shell HS, were very similar in all eggshells. In contrast, the composition of calcified shell CS disaccharides was highly variable. In membranes, both HA and KS content showed a correlation with egg shape index. The 4SCS-A content correlated with eggshell strength, and 0SCS-0 correlated with eggshell strength and calcified shell thickness. HS content and its disaccharide composition showed no apparent correlation to properties of calcified shells. In calcified shells, only HS 6S correlated with egg shape index. This study suggests that GAG content and disaccharide composition of shell membranes might impact the quality of chicken eggshells.

The gentio-oligosaccharide gentiobiose functions in the modulation of bud dormancy in the herbaceous perennial Gentiana.

Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [ß-D-Glcp-(1→6)-D-Glc] and gentianose [ß-D-Glcp-(1→6)-D-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of ß-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.

Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept.

Plants suffering from abiotic stress are commonly facing an enhanced accumulation of reactive oxygen species (ROS) with damaging as well as signalling effects at organellar and cellular levels. The outcome of an environmental challenge highly depends on the delicate balance between ROS production and scavenging by both enzymatic and metabolic antioxidants. However, this traditional classification is in need of renewal and reform, as it is becoming increasingly clear that soluble sugars such as disaccharides, raffinose family oligosaccharides and fructans--next to their associated metabolic enzymes--are strongly related to stress-induced ROS accumulation in plants. Therefore, this review aims at extending the current concept of antioxidants functioning during abiotic stress, with special focus on the emanate role of sugars as true ROS scavengers. Examples are given based on their cellular location, as different organelles seem to exploit distinct mechanisms. Moreover, the vacuole comes into the picture as important player in the ROS signalling network of plants. Elucidating the interplay between the mechanisms controlling ROS signalling during abiotic stress will facilitate the development of strategies to enhance crop tolerance to stressful environmental conditions.

Sensing UV/blue: pterin as a UV-A absorbing chromophore of cryptochrome.

Cyanobacteria sense and respond to changes in an ambient light environment using highly specialized photoreceptors coupled to signal transduction pathways. Synechocystis sp. PCC 6803 is currently used as a model system to study light signal transduction in cyanobacteria. Recently, several important players, including photoreceptors and other signaling partners, have been identified in Synechocystis sp. PCC 6803. However, the nature of the molecules that act as UV/blue light sensors (and their downstream signaling mechanism) has not been elucidated. It has been postulated that pterins might serve as possible photoreceptor pigments for some behavioral responses induced by UV/blue light. By investigating the photomovement of wild-type and a pgtA mutant to UV/blue light, we demonstrated that cyanopterin is indeed involved in inhibiting negative phototaxis under UV/blue light. In this addendum, we provide additional evidence showing that the UV/blue action spectrum of the phototactic response coincides with the fluorescence spectrum of the in vivo cyanobacterial cryptochrome, DASH. Based on these results, we discuss the potential role of pterin as a UV-A absorbing chromophore of the cryptochrome in Synechocystis sp. PCC 6803.

Immune modulation by chondroitin sulfate and its degraded disaccharide product in the development of an experimental model of multiple sclerosis.

Clinical symptoms in MOG-induced EAE mice significantly exacerbated following chondroitin sulfate A (CS-A) injection, whereas administration of a degraded product, CSPG-DS, caused dramatic inhibition of EAE development. Also, administration of CSPG-DS but not CS-A, after the onset of clinical symptoms of EAE, was able to suppress the disease. Further studies demonstrated that CS-A up-regulated STAT4 expression and thus, induced IFN-gamma production and Th1 CD4 T cell differentiation. CS-A also up-regulated STAT3 and IL-23 expression and thus increased IL-17 producing T cells. CSPG-DS treatment both in vivo and in vitro decreased TNFalpha production from splenocytes. In vitro and in vivo studies indicated that CSPG-DS treatment in EAE mice significantly blocked migration of lymphocytes, whereas CS-A treatment increased lymphocyte infiltration in the brain.

Alpha-D-mannopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-glycerate in the thermophilic bacterium Petrotoga miotherma--structure, cellular content and function.

The intracellular accumulation of low molecular mass organic compounds in response to stressful conditions was investigated in the thermophilic bacterium Petrotoga miotherma, a member of the order Thermotogales. This led to the discovery of a new solute, whose structure was established as alpha-D-mannopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-glycerate (MGG) by MMR spectroscopy and MS. Under optimum growth conditions (3% NaCl; 55 degrees C), MGG was the major solute [up to 0.6 micromol.(mg protein)(-1)]; alpha-glutamate and proline were also present but in minor amounts [below 0.08 micromol.(mg protein)(-1)]. The level of MGG increased notably with the salinity of the growth medium up to the optimum NaCl concentration. At higher NaCl concentrations, however, the level of MGG decreased, whereas the levels of proline and alpha-glutamate increased about five-fold and 10-fold, respectively. MGG plays a role during low-level osmotic adaptation of Petrotoga miotherma, whereas alpha-glutamate and, to a lesser extent, proline are used for osmoprotection under salt stress. MGG is not part of the cell strategy for coping with heat or oxidative stress. Nevertheless, MGG was an efficient protector of pig heart malate dehydrogenase against heat inactivation and freeze-drying, although mannosylglycerate was better. This is the first report on the occurrence of MGG in living systems.

Foundations of antibiotic resistance in bacterial physiology: the mycobacterial paradigm.

The intrinsic resistance of Mycobacterium tuberculosis and related pathogens to most common antibiotics limits chemotherapeutic options to treat tuberculosis and other mycobacterial diseases. Resistance has traditionally been attributed to the unusual multi-layer cell envelope that functions as an effective barrier to the penetration of antibiotics. Recent insights into mechanisms that neutralize the toxicity of antibiotics in the cytoplasm have revealed systems that function in synergy with the permeability barrier to provide intrinsic resistance. Here, we highlight the growing pool of information about internal, antibiotic-responsive regulatory proteins and corresponding resistance genes, and present new concepts that rationalize how they might have evolved. Pharmaceutical inhibition of these intrinsic systems could make many previously available antibiotics active against M. tuberculosis.

No role of alpha-Gal in human monocyte-endothelial cell interactions in vitro.

Vascularized organ xenografts undergoing acute vascular rejection (AVR) are infiltrated by innate immune cells such as monocytes/macrophages. Herein, human monocyte static and dynamic adhesion to, and migration across, human and porcine aortic endothelial cells (HAEC and PAEC) were investigated. To elucidate the role of Gal alpha1,3Gal (alpha-Gal) epitopes in these processes in the absence of anti-Gal antibodies (Ab), this determinant was aberrantly expressed in HAEC. HAEC were transduced with a lentiviral vector encoding the porcine alpha1,3 galactosyltransferase to express alpha-Gal at high frequencies (75-95%). Alpha-Gal expression on HAEC did not increase their ability to support monocyte transendothelial migration or adhesion under either static or flow conditions. Porcine and human endothelium supported static adhesion and migration of monocytes equally well. However, human monocytes adhered less to PAEC than to HAEC (P = 0.03) under flow following human, but not porcine, tumour necrosis factor-alpha stimulation. In the absence of anti-Gal Ab, the alpha-Gal epitope does not contribute to increased monocyte adhesion to, or migration across, endothelium. Thus, inhibiting adhesion receptor-ligand interactions essential for the adhesion of human monocytes to porcine endothelium may be more important than carbohydrate remodelling of donor pigs to prevent adhesion/infiltration of monocytes into organ xenografts during AVR.

Monocyte adhesion to xenogeneic endothelium during laminar flow is dependent on alpha-Gal-mediated monocyte activation.

Monocytes are the predominant inflammatory cell recruited to xenografts and participate in delayed xenograft rejection. In contrast to allogeneic leukocytes that require up-regulation of endothelial adhesion molecules to adhere and emigrate into effector tissues, we demonstrate that human monocytes adhere rapidly to unstimulated xenogeneic endothelial cells. The major xenoantigen galactosealpha(1,3)galactosebeta(1,4)GlcNAc-R (alpha-gal) is abundantly expressed on xenogeneic endothelium. We have identified a putative receptor for alpha-gal on human monocytes that is a member of the C-type family of lectin receptors. Monocyte arrest under physiological flow conditions is regulated by alpha-gal, because cleavage or blockade results in a dramatic reduction in monocyte adhesion. Recruitment of human monocytes to unactivated xenogeneic endothelial cells requires both alpha(4) and beta(2) integrins on the monocyte; binding of alpha-gal to monocytes results in rapid activation of beta(2), but not alpha(4), integrins. Thus, activation of monocyte beta(2) integrins by alpha-gal expressed on xenogeneic endothelium provides a mechanism that may explain the dramatic accumulation of monocytes in vivo.

Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetylgalactosaminyltransferase and alpha1,3-fucosyltransferase.

Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAcbeta1-4GlcNAcbeta1-)n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Galbeta1-4GlcNAcbeta1-)n. We also stably expressed human alpha1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha1,3-fucosylated LDN (LDNF) GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-R as well as novel "poly-LDNF" structures (-3GalNAcbeta1-4(Fucalpha 1-3)GlcNAcbeta1-)n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAcbeta1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.

Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells.

Galactose alpha1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process.

Reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human CD55 or deficient for the galactosyl-alpha(1-3) galactosyl epitope.

Complement activation mediated by the major xenogeneic epitope in the pig, galactosyl-alpha(1-3) galactosyl sugar structure (alpha-Gal), and human natural antibodies could cause hyperacute rejection (HAR) in pig-to-human xenotransplantation. The same reaction on viruses bearing alpha-Gal may serve as a barrier to zoonotic infection. Expressing human complement regulatory proteins or knocking out alpha-Gal epitopes in pig in order to overcome HAR may therefore pose an increased risk in xenotransplantation with regard to zoonosis. We investigated whether amphotropic murine leukemia virus, porcine endogenous retrovirus, and vesicular stomatitis virus (VSV) budding from primary transgenic pig aortic endothelial (TgPAE) cells expressing human CD55 (hCD55 or hDAF) was protected from human-complement-mediated inactivation. VSV propagated through the ST-IOWA pig cell line, in which alpha-galactosyl-transferase genes were disrupted (Gal null), was also tested for sensitivity to human complement. The TgPAE cells were positive for hCD55, and all pig cells except the Gal-null ST-IOWA expressed alpha-Gal epitopes. Through antibody binding, we were able to demonstrate the incorporation of hCD55 onto VSV particles. Viruses harvested from TgPAE cells were relatively resistant to complement-mediated inactivation by the three sources of human sera tested. Additionally, VSV from Gal-null pig cells was resistant to human complement inactivation. Such protection of enveloped viruses may increase the risk of zoonosis from pigs genetically modified for pig-to-human xenotransplantation.

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion.

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.

Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics.

Mycothiol, MSH or 1D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, is an unusual conjugate of N-acetylcysteine (AcCys) with 1D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins), and is the major low-molecular-mass thiol in mycobacteria. Mycothiol has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. Because of these activities, MSH is a candidate for protecting Mycobacterium tuberculosis from inactivation by the host during infections as well as for resisting antituberculosis drugs. In order to define the protective role of MSH for M. tuberculosis, we have constructed an M. tuberculosis mutant in Rv1170, one of the candidate MSH biosynthetic genes. During exponential growth, the Rv1170 mutant bacteria produced approximately 20% of wild-type levels of MSH. Levels of the Rv1170 substrate, GlcNAc-Ins, were elevated, whereas those of the product, GlcN-Ins, were reduced. This establishes that the Rv1170 gene encodes for the major GlcNAc-Ins deacetylase activity (termed MshB) in the MSH biosynthetic pathway of M. tuberculosis. The Rv1170 mutant grew poorly on agar media lacking catalase and oleic acid, and had heightened sensitivities to the toxic oxidant cumene hydroperoxide and to the antibiotic rifampin. In addition, the mutant was more resistant to isoniazid, suggesting a role for MSH in activation of this prodrug. These data indicate that MSH contributes to the protection of M. tuberculosis from oxidants and influences resistance to two first-line antituberculosis drugs.

Rapid static adhesion of human naïve neutrophil to naïve xenoendothelium under physiologic flow is independent of Galalpha1,3-gal structures.

Adhesion interactions under flow have long been known to depend on applied wall shear stress. We investigated the ability of human naïve neutrophils to adhere to xenogeneic endothelial cells under static and flow conditions. We demonstrate that human naïve neutrophils bind to xenogeneic endothelial cells under flow conditions. This binding is dependent on the applied stress and is independent of Galalpha1,3-gal structures, ICAM-1, or its counter ligands LFA-1alpha and Mac-1. The binding was rapid and is characterized by stationary attachment with no obvious rolling or change in morphology. This binding leads to a transient increase in intracellular-free calcium levels in xenogeneic but not allogeneic-endothelial cells with occasional oscillations that persist long after the initial contact between the two cell types. Previous activation of xenoendothelium by autologous serum or human TNF-alpha augments binding of human naïve neutrophils to the endothelial cells. Our data suggest novel interaction sites between the xenogeneic endothelial cells and human naïve neutrophils.