Carbohydrate hitched imidazoles as agents for the disruption of fungal cell membrane.

The fungal diseases represent an increasing global health burden and have transformed from a rare curiosity to the leading cause of human mortality. The present manuscript reports the antifungal potential of two novel compounds possessing a carbohydrate and an imidazole moiety. Antifungal susceptibility test determined the growth inhibition potential of the synthesized compounds against Aspergillus niger 9689 and it was observed that compounds D and E gave an antifungal inhibitory index of 66.66 and 56.67% respectively. Further, ultra-structure analysis of the treated fungal mycelia through scanning electron microscope (SEM) and confocal microscopy indicated significant membrane permeability and disintegration of fungal cell membrane, thus highlighting the probable role of the synthesized compounds as inhibitors of fungal lanosterol 14α-demethylase. In silico studies corroborated with the in-vitro results, as the synthesized compounds interacted with the critical amino acids present at the active site of the fungal enzyme (lanosterol 14α-demethylase).

Function and structure of GH13_31 α-glucosidase with high α-(1→4)-glucosidic linkage specificity and transglucosylation activity.

α-Glucosidase hydrolyzes α-glucosides and transfers α-glucosyl residues to an acceptor through transglucosylation. In this study, GH13_31 α-glucosidase BspAG13_31A with high transglucosylation activity is reported in Bacillus sp. AHU2216 and biochemically and structurally characterized. This enzyme is specific to α-(1→4)-glucosidic linkage as substrates and transglucosylation products. Maltose is the most preferred substrate. Crystal structures of BspAG13_31A wild-type for the substrate-free form and inactive acid/base mutant E256Q in complexes with maltooligosaccharides were solved at 1.6-2.5 Å resolution. BspAG13_31A has a catalytic domain folded by an (ß/α)8 -barrel. In subsite +1, Ala200 and His203 on ß→α loop 4 and Asn258 on ß→α loop 5 are involved in the recognition of maltooligosaccharides. Structural basis for specificity of GH13_31 enzymes to α-(1→4)-glucosidic linkage is first described.

Conserved FcγR- glycan discriminates between fucosylated and afucosylated IgG in humans and mice.

The binding strength between IgG and FcγR is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human FcγRIIIa up to ∼20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to ∼40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate-carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFcγRIIIa and hFcγRIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse FcγR, mFcγRIV. The N162-glycan in mFcγRIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFcγRIIIa. However, unlike hFcγRIIIa, mFcγRIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human FcγRIII family and its orthologous mouse FcγRIV to be functionally conserved.

N-linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens.

BACKGROUND: The temporary or long-term xenotransplantation of pig organs into people would save thousands of lives each year if not for the robust human antibody response to pig carbohydrates. Genetically engineered pigs deficient in galactose α1,3 galactose (gene modified: GGTA1) and N-glycolylneuraminic acid (gene modified: CMAH) have significantly improved cell survival when challenged by human antibody and complement in vitro. There remains, however, a significant portion of human antibody binding. METHODS: To uncover additional xenoantigens, we compared the asparagine-linked (N-linked) glycome from serum proteins of humans, domestic pigs, GGTA1 knockout pigs, and GGTA1/CMAH knockout pigs using mass spectrometry. Carbohydrate structures were determined with assistance from GlycoWorkbench, Cartoonist, and SimGlycan software by comparison to existing database entries and collision-induced dissociation fragmentation data. RESULTS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of reduced and solid-phase permethylated glycans resulted in the detection of high-mannose, hybrid, and complex type N-linked glycans in the 1000-4500 m/z ion range. GGTA1/CMAH knockout pig samples had increased relative amounts of high-mannose, incomplete, and xylosylated N-linked glycans. All pig samples had significantly higher amounts of core and possibly antennae fucosylation. CONCLUSIONS: We provide for the first time a comparison of the serum protein glycomes of the human, domestic pig, and genetically modified pigs important to xenotransplantation.

α1,3Galactosyltransferase knockout pigs produce the natural anti-Gal antibody and simulate the evolutionary appearance of this antibody in primates.

BACKGROUND: Anti-Gal is the most abundant natural antibody in humans and Old World primates (apes and Old World monkeys). Its ligand, the α-gal epitope (Galα1-3Galß1-4GlcNAc-R), is abundant in nonprimate mammals, prosimians and New World monkeys whereas it is absent in humans and Old World primates as a result of inactivation of the α1,3galactosyltransferase (α1,3GT) gene in ancestral Old World primates, as recent as 20-28 million years ago. Since anti-Gal has been a "forbidden" autoantibody for >140 million years of evolution in mammals producing α-gal epitopes it was of interest to determine whether ancestral Old World primates could produce anti-Gal once α-gal epitopes were eliminated, i.e. did they carry anti-Gal encoding immunoglobulin genes, or did evolutionary selection eliminate these genes that may be detrimental in mammals synthesizing α-gal epitopes. This question was studied by evaluating anti-Gal prodution in α1,3GT knockout (GT-KO) pigs recently generated from wild-type pigs in which the α-gal epitope is a major self-antigen. METHODS: Anti-Gal antibody activity in pig sera was assessed by ELISA, flow cytometry and complement mediated cytolysis and compared to that in human sera. RESULTS: The study demonstrates abundant production of the natural anti-Gal antibody in GT-KO pigs at titers even higher than in humans. The fine specificity of GT-KO pig anti-Gal is identical to that of human anti-Gal. CONCLUSIONS: Pigs and probably other mammals producing α-gal epitopes carry immunoglobulin genes encoding anti-Gal as an autoantibody. Once the α-gal epitope is eliminated in GT-KO pigs, they produce anti-Gal. These findings strongly suggest that similar to GT-KO pigs, inactivation of the α1,3GT gene in ancestral Old World primates enabled the immediate production of anti-Gal, possibly as a protective antibody against detrimental microbial agents carrying α-gal epitopes.

Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells.

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4  pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1  ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4  ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.

Chemical and biological properties of a highly branched ß-glucan from edible mushroom Pleurotus sajor-caju.

Hot aqueous extraction of the basidiocarps of the mushroom Pleurotus sajor-caju provided a cold water-soluble, gel-like glucan, which was characterized chemically, and its effects on RAW 264.7 cell line (mouse leukaemic monocyte macrophage) activation were determined. NMR spectroscopy, HPSEC, methylation analysis, and a controlled Smith degradation showed it to have a branched structure with a (1→3)-linked ß-Glcp main-chain, substituted at O-6 by single-unit ß-Glcp side-chains, on the average of two to every third residues of the backbone, with a molar mass of 9.75 × 10(5) g mol(-1). In macrophage cell culture, the ß-glucan induced production of NO and the cytokines TNF-α, IL-1ß, these effects being very similar as those of Escherichia coli serotype 0111:B4 Sigma-Aldrich lipopolysaccharide (LPS), although not modifying the response of LPS-activated macrophages. The results suggest that the (1→3), (1→6)-linked ß-glucan from P. sajor-caju may have potential for immunological activities, although additional experiments are necessary for a better understanding of the mechanisms involved.

Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability.

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

Glycome-DB.org: a portal for querying across the digital world of carbohydrate sequences.

Despite ongoing harmonization efforts, the major carbohydrate sequence databases following the first initiative in this field, CarbBank, are still isolated islands, with mechanisms for automatic structure exchange and comparison largely missing. This unfavorable situation has been overcome with a systematic data integration effort, resulting in the GlycomeDB, a meta-database for public carbohydrate sequences. It contains at present 35,056 unique structures in GlycoCT encoding, referencing more than 100,000 external records from 1845 different taxonomic sources. We have created a user-friendly, web-based graphical interface which allows taxonomic and structural data to be entered and searched for. The structural search possibilities include substructure search, similarity search, and maximum common substructure. A novel search refinement mechanism allows the assembly of complex queries. With GlycomeDB (www.glycome-db.org), it is now possible to use a single portal to access all digitally encoded, public structural data in glycomics and to perform complex queries with the help of a web-based user interface.

Molecular structure of heparan sulfate from Spalax. Implications of heparanase and hypoxia.

Spalax, a subterranean blind mole rat, is well adapted to live in an extreme hypoxic environment through up-regulated expression of growth factors and enzymes for ensuring sufficient oxygen supply. One of the overexpressed enzymes is heparanase, an endoglucuronidase that selectively cleaves heparan sulfate (HS) and is implicated in angiogenesis. To assess the implications of the heparanase in Spalax, we have characterized the structure of HS isolated from various organs of the animal. The oligosaccharides obtained after deaminative cleavage of HS samples from the tissues show an overall higher sulfation degree, distinct from that of murine tissues. Of particular significance was the appearance of a trisaccharide moiety in the tissues examined, apart of the even numbered oligosaccharide fractions typically found in HS from human and mouse tissues. The formation of this odd-numbered saccharide is a consequence of heparanase action, in agreement with the notion of high expression of the enzyme in this species. Analysis of HS extracted from human embryonic kidney cells (HEK293) after exposure to hypoxic condition revealed a structural change in the distribution of oligosaccharides similar to HS derived from Spalax organs. The alterations are likely due to up-regulated activity of heparanase, as real-time RT-PCR showed a 2-fold increase in heparanase mRNA expression in the hypoxia treated cells. HEK293 cells stably overexpressing Spalax heparanase produced HS sharing similarity with that from the Spalax organs, and exhibited enhanced MAPK activity in comparison with HEK293 cells, indicating a regulation role of the heparanase in the activity of growth factors.

Non-anticoagulant heparins and inhibition of cancer.

Low-molecular-weight heparins (LMWH) appear to prolong survival of patients with cancer. Such a beneficial effect is thought to be associated with interruption of molecular mechanisms involving the heparan sulfate (HS) chains of cell surface and extracellular matrix proteoglycans (HSPGs), growth factors and their receptors, heparanase, and selectins. The beneficial effects of heparin species could also be associated with their ability to release tissue factor pathway inhibitor from endothelium. The utility of heparin and LMWH as anticancer drugs is limited due to their anticoagulant properties. Non-anticoagulant heparins can be obtained either by removing chains containing the antithrombin-binding sequence, or by inactivating critical functional groups or units of this sequence. The non-anticoagulant heparins most extensively studied are regioselectively desulfated heparins and 'glycol-split' heparins. Some modified heparins of both types are potent inhibitors of heparanase. A number of them also attenuate metastasis in experimental models. With cancer cells overexpressing selectins, heparin-mediated inhibition of tumor cells-platelets aggregation and tumor cell interaction with the vascular endothelium appears to be the prevalent mechanism of attenuation of early stages of metastasis. The structural requirements for inhibition of growth factors, heparanase, and selectins by heparin derivatives are somewhat different for the different activities. An N-acetylated, glycol-split heparin provides an example of application of a non-anticoagulant heparin that inhibits cancer in animal models without unwanted side effects. Delivery of this compound to mice bearing established myeloma tumors dramatically blocked tumor growth and progression.

[Proteus bacilli: features and virulence factors]. / Bakterie z rodzaju Proteus--cechy i czynniki chorobotwórczosci.

In this article, different aspects of virulence factors of Proteus bacilii (P. mirabilis, P. vulgaris, P. penneri i P. hauseri) are presented. These are opportunistic pathogens that cause different kinds of infections, most frequently of the urinary tract. These bacteria have developed several virulence factors, such as adherence due to the presence of fimbriae or afimbrial adhesins, invasiveness, swarming phenomenon, hemolytic activity, urea hydrolysis, proteolysis, and endotoxicity. Below we focus on data concerning the molecular basis of the pathogenicity of Proteus bacilli.

Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures.

It is known that heterogeneous conditions exist in large-scale animal cell cultures. However, little is known about how heterogeneities affect cells, productivities, and product quality. To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator. Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation. Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales. Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures. Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes. Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield. DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb). The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature. However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody. It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb. Such results underline the importance of monitoring protein characteristics for the development of large-scale processes.

Evidence of block and randomly sequenced chondroitin polysaccharides: sequential enzymatic digestion and quantification using ion trap tandem mass spectrometry.

A method for determining the sequence type of the disaccharide repeat region of cartilage samples is introduced. The samples are sequentially subjected to selective and nonselective enzymatic digestion, and the isomeric products from each step are quantified using tandem mass spectrometry. The two-step digestion/quantification protocol identifies whether the global makeup of the polymer is "alternating", "random", or "blocked" with respect to the two main components of the cartilage, 4- and 6-sulfated disaccharides. Using this procedure, the sequence type of two biologically isolated chondroitin polysaccharides was identified. The results for chondroitin sulfate A, isolated from bovine trachea, are consistent with the 4- and 6-sulfated disaccharides randomly distributed throughout the repeat region of the polysaccharide. For chondroitin sulfate C, shark cartilage, the 6-sulfated disaccharides are adjacent to each other to a larger extent than one would expect for a randomly distributed polymer, indicating that "blocks" of repeating disaccharides with the same sulfation site are present.

Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding.

Several lines of evidence indicate that mammalian fertilization is initiated via a binding process that is dependent upon the recognition of oligosaccharide sequences associated with zona pellucida (ZP) glycoproteins. Here, specific chemical and enzymatic methods were employed to modify human ZP and to test their effects on sperm binding in the hemizona assay system (HZA). Periodate oxidation of human ZP under very mild conditions (10 min, 0 degrees C, 1 mM sodium m-periodate) that attacks only terminal sialic acid resulted in a 30% loss of human sperm binding in the HZA [hemizona index (HZI) = 70.2 +/- 10.9, n = 22; P < 0.05]. Periodate oxidation under mild conditions (1 h, 23 degrees C, 10 mM sodium m-periodate) caused a 40% decrease in binding (HZI = 60.8 +/- 10.3; n = 24; P< 0.01). Treatment of human ZP with neuraminidase caused a substantial increase in sperm binding to human ZP (HZI = 297 +/- 45, n = 22; P < 0.01). These findings indicate that there are sialic acid dependent binding sites coexisting with binding sites that are obscured by sialic acid. To determine the periodate sensitivity of these obscured sites, hemizona were first digested with neuraminidase and subsequently subjected to mild periodate oxidation. The combined enzymatic and chemical treatments caused a 79% decrease in sperm binding compared to control hemizona (HZI = 20.7 +/- 4.4, n = 16; P < 0.001). Human sperm-ZP interaction was also increased by digestion of human ZP with endo-beta-galactosidase (HZI = 710 +/- 232, n = 14; P < 0.01), indicating that potential binding sites for spermatozoa are also obscured by lactosaminoglycan sequences. These studies support a definitive role for the involvement of ZP-associated glycans in the binding of human spermatozoa to oocytes.