Biochemical characterization of Helicobacter pylori α-1,4 fucosyltransferase: metal ion requirement, donor substrate specificity and organic solvent stability

The effect of metal ions on the activity, the donor substrate specificity, and the stability in organic solvents of Helicobacter pylori α-1,4 fucosyltransferase were studied. The recombinant enzyme was expressed as soluble form in E. coli strain AD494 and purified in a one step affinity chromatography. Its activity was highest in cacodylate buffer at pH 6.5 in the presence of 20mM Mn2+ ions at 37°C. Mn2+ ions could be substituted by other metal ions. In all cases, Mn2+ ions proofed to be the most effective (Mn2+>Co2+>Ca2+>Mg2+>Cu2+>Ni2+>EDTA). The enzyme shows substrate specificity for Type I disaccharide (1) with a K M of 114μM. In addition, the H. pylori α-1,4 fucosyltransferase efficiently transfers GDP-activated l-fucose derivatives to Galβ1-3GlcNAc-OR (1). Interestingly, the presence of organic solvents such as DMSO and methanol up to 20% in the reaction medium does not affect significantly the enzyme activity. However, at the same concentration of dioxane, activity is totally abolishe

2-C-Branced mannosides as a novel family of FimH antagonists - Synthesis and biological evaluation

Urinary tract infections (UTIs), which are among the most prevalent bacterial infections worldwide, are mainly attributed to uropathogenic Escherichia coli (UPEC). Because of frequent antibiotic treatment, antimicrobial resistance constitutes an increasing therapeutic problem. Antagonists of the mannose-specific bacterial lectin FimH, a key protein mediating the adhesion of UPEC to human bladder cells, would offer an alternative anti-adhesive treatment strategy. In general, FimH antagonists consist of a mannose moiety and a wide range of lipophilic aglycones. Modifications of the mannose core led to a distinct drop in affinity. A visual inspection of the crystal structure of FimH revealed a previously unexplored cavity surrounded by Ile13, Phe142 and Asp140, which could be reached by functional groups in the equatorial 2-position of the mannose. Here, we describe the synthesis of 2- C -branched mannosides and evaluation of their pharmacodynamic properties. ITC experiments with the selected antagonists revealed a drastic enthalpy loss for all 2- C -branched antagonists, which, however, is partially compensated by an entropy gain. This supports the hypothesis that the target cavity is too small to accommodate 2- C -substituents

Molecular cloning and functional expression of a novel Helicobacter pylori α-1,4 fucosyltransferase

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is associated with gastric cancer and lymphoma. This microorganism synthesizes fucosylated oligosaccharides, predominantly the Galb-1,4GlcNAc (Type II) blood group antigens Lewis X and Y, whereas a small population also expresses the Galb-1,3GlcNAc (Type I) blood group antigens Lewis A and B. These carbohydrate structures are known to mimic host cell antigens and permit the bacteria to escape from the host immune response. Here, we report the cloning and characterization of a novel H. pylori α-1,4 fucosyltransferase (FucT). In contrast to the family members characterized to date, this enzyme shows exclusively Type I acceptor substrate specificity. The enzyme consisting of 432 amino acids (MW 50,502 Da) was cloned using a polymerase chain reaction (PCR)-based approach. It exhibits a high degree of identity (75-87%) and similar structural features, for example, in the heptamer repeat pattern, with other H. pylori FucTs. The kinetic characterization revealed a very efficient transferase (kcat/Km = 229 mM21s21) for the Type I acceptor substrate (Gal)-1,3 GlcNAc-Lem (1). Additionally, the enzyme possesses a broad tolerance toward nonnatural Type I acceptor substrate analogs and therefore represents a valuable tool for the chemoenzymatic synthesis of Lewis A, sialyl Lewis A as well as mimetics thereo

Implications of the E-selectin S128R mutation for drug discovery

The C-type lectin E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells during the inflammatory process. In numerous studies, the S128R mutation of the E-selectin was associated with cardiovascular and autoimmune diseases. There is evidence that the S128R E-selectin mutation leads to a loss in ligand specificity, thus increasing leukocyte recruitment. Apart from the natural tetrasaccharide ligand sialyl Lewisx (sLex), it has previously been proposed that non-fucosylated carbohydrates also bind to S128R E-selectin. To evaluate the therapeutic potential of the antagonism of the E-selectin mutant, ligand specificity was reinvestigated on a molecular basis. We determined the ligand specificity of wild-type and S128R E-selectin in a target-based competitive assay, a glycan array screen and cell-based binding assays under static and flow conditions. Regarding ligand-specificity, the binding properties of S128R E-selectin were identical to those of wt E-selectin, i.e., no mutant-specific binding of 3′-sialyl-N-acetyllactosamine, heparin, fetuin and K562 cells was observed. Additionally, the binding affinities of glycomimetic E-selectin antagonists were identical for wt and S128R E-selectin. Overall, the previous reports on carbohydrate ligand promiscuity of S128R E-selectin could not be confirme

Magnetic resonance imaging in the investigation of canine heads

A imagem por ressonância magnética (IRM) é o método de diagnóstico por imagem não invasivo mais sensível para avaliar as partes moles, particularmente o encéfalo, porém trata-se de uma técnica onerosa. O método fundamenta-se no fenômeno da ressonância magnética nuclear que ocorre quando núcleos atômicos com propriedades magnéticas presentes no corpo são submetidos a um campo magnético intenso, sendo posteriormente excitados por energia de radiofrequência e gerando, por sua vez, um sinal de onda de radiofrequência capaz de ser captado por uma antena receptora, passando por um processo matemático, chamado Transformada de Fourier, para posterior formação da imagem. Esse estudo objetivou realizar 10 exames completos da cabeça em cadáveres de cães normais à IRM e confeccionar um Atlas com as estruturas identificadas. As imagens foram adquiridas em um aparelho de ressonância magnética Gyroscan S15/HP Philips com campo magnético de 1,5Tesla. Os cadáveres foram posicionados com a cabeça no interior de uma bobina de cabeça humana e foram submetidos a cortes iniciais sagitais a partir de onde se planejou os cortes transversais e dorsais nas sequências de pulso spin-eco T1, T2 e DP. Em T1 utilizou-se TR=400ms e TE=30ms, T2 utilizou-se TR=2000ms e TE=80ms e na DP utilizou-se TR=2000ms e TE=30ms. A espessura do corte foi de 4mm, o número de médias foi igual a 2, a matriz foi de 256x256, o fator foi igual a 1,0 e o campo de visão foi de 14cm. A duração do exame completo da cabeça foi de 74,5minutos. As imagens obtidas com as sequências utilizadas e com a bobina de cabeça humana foram de boa qualidade. Em T1 a gordura tornou-se hiperintensa e o líquido hipointenso. Em T2 a gordura ficou menos hiperintensa e o líquido hiperintenso. A cortical óssea e o ar foram hipointensos em todas as sequências utilizadas devido a baixa densidade de prótons. A sequência DP mostrou o melhor contraste entre a substância branca e cinzenta quando comparada a T2 e a T1. T2 evidenciou o líquido cefalorraquidiano tornando possível a distinção dos sulcos e giros cerebrais. Através do exame de IRM foi possível, pelo contraste, identificar as estruturas ósseas componentes da arquitetura da região, músculos, grandes vasos venosos e arteriais e estruturas do sistema nervoso central, além de elementos do sistema digestório, respiratório e estruturas dos olhos entre outras. Nesse estudo as IRM adquiridas nas sequências T1, DP e T2 foram complementares para o estudo dos aspectos anatômicos da cabeça de cães demonstrando-os com riqueza de detalhes. O tempo requerido para o exame completo da cabeça é compátivel para uso em animais vivos desde que devidamente anestesiados e controlados. Os resultados obtidos por esse trabalho abrem caminho em nosso meio, para o estudo de animais vivos e para o início da investigação de doenças, principalmente as de origem neurológica, visto ser esta técnica excelente para a visibilização do encéfalo.Magnetic resonance imaging (MRI) is the most sensitive method of diagnostic imaging to evaluate soft tissues, specially the brain, however it is expensive. The method is based on the nuclear magnetic resonance phenomenon that occurs when atomic nucleus with magnetic proprieties in the body are submitted to a strong magnetic field, and excited with radio frequency generating a radio frequency signal captured by a receptive antenna. The signal is processed by Fourier Transform for the image formation. This study had the objective to obtain 10 complete exams of heads in cadavers of normal dogs to MRI and to make an Atlas of head structures. The images were obtained with a magnetic resonance unit Gyroscan S15/HP Philips using a magnetic field of 1,5Tesla. The cadavers were positioned with the head into a human head coil and submitted to sagittal slices used to plan transverse and dorsal slices in T1, T2 and DP spin-echo sequences. In T1 we adjusted TR=400ms and TE=30ms, in T2 TR=2000ms and TE=80ms and in DP TR=2000ms and TE=30ms. The slice thickness was 4mm, the number of averages 2, the matrix 256x256, the factor 1,0 and the field of view 14cm. The duration of the complete exam of the head was 74,5minutes. The images obtained with the described sequences and with the human head coil was of good quality. In T1 fat was hyperintense and fluid was hypointense. In T2 fat was less hyperintense and fluid was hyperintense. The cortical bone and the air were hypointense in all sequences used because of the low proton density. The DP sequence showed the best contrast between white and gray matter when compared with T2 and T1 sequences. Distinction of cerebral sulcus and gyrus was possible because T2 showed the cerebrospinal fluid. The identification of bone structures that compound the region, muscles, main venous and arterial vessels and structures of the central nervous system, besides elements of the digestory and respiratory systems and structures of the eyes among others was possible through contrast obtained with MRI. In this study the MRI acquired in T1, DP and T2 were complementary for the anatomic study of the head and been able to demonstrate the structures of the canine head with rich anatomic details. The time used to do the complete exam of the head is compatible with the use in live animals since properly anesthetized and controlled. We had opened a way for the study of live animals and for the beginning of disease investigation, mainly that of neurologic origin because this technique is excellent for brain visualization

Glucose-induced down regulation of thiamine transporters in the kidney proximal tubular epithelium produces thiamine insufficiency in diabetes

Increased renal clearance of thiamine (vitamin B1) occurs in experimental and clinical diabetes producing thiamine insufficiency mediated by impaired tubular re-uptake and linked to the development of diabetic nephropathy. We studied the mechanism of impaired renal re-uptake of thiamine in diabetes. Expression of thiamine transporter proteins THTR-1 and THTR-2 in normal human kidney sections examined by immunohistochemistry showed intense polarised staining of the apical, luminal membranes in proximal tubules for THTR-1 and THTR-2 of the cortex and uniform, diffuse staining throughout cells of the collecting duct for THTR-1 and THTR-2 of the medulla. Human primary proximal tubule epithelial cells were incubated with low and high glucose concentration, 5 and 26 mmol/l, respectively. In high glucose concentration there was decreased expression of THTR-1 and THTR-2 (transporter mRNA: −76% and −53% respectively, p<0.001; transporter protein −77% and −83% respectively, p<0.05), concomitant with decreased expression of transcription factor specificity protein-1. High glucose concentration also produced a 37% decrease in apical to basolateral transport of thiamine transport across cell monolayers. Intensification of glycemic control corrected increased fractional excretion of thiamine in experimental diabetes. We conclude that glucose-induced decreased expression of thiamine transporters in the tubular epithelium may mediate renal mishandling of thiamine in diabetes. This is a novel mechanism of thiamine insufficiency linked to diabetic nephropathy

Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding

The most prevalent diseases manifested by Escherichia coli are acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn's disease. E. coli clinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connected via a pilin domain to the tip of type 1 pili. Although the isolated FimH lectin domain has affinities in the nanomolar range for all high-mannosidic glycans, differentiation between these glycans is based on their capacity to form predominantly hydrophobic interactions within the tyrosine gate at the entrance to the binding pocket. In this study, novel crystal structures of tyrosine-gate mutants of FimH, ligand-free or in complex with heptyl α - D - O -mannopyranoside or 4-biphenyl α - D - O- mannopyranoside, are combined with quantum-mechanical calculations and molecular-dynamics simulations. In the Y48A FimH crystal structure, a large increase in the dynamics of the alkyl chain of heptyl α - D - O -mannopyranoside attempts to compensate for the absence of the aromatic ring; however, the highly energetic and stringent mannose-binding pocket of wild-type FimH is largely maintained. The Y137A mutation, on the other hand, is the most detrimental to FimH affinity and specificity: (i) in the absence of ligand the FimH C-terminal residue Thr158 intrudes into the mannose-binding pocket and (ii) ethylenediaminetetraacetic acid interacts strongly with Glu50, Thr53 and Asn136, in spite of multiple dialysis and purification steps. Upon mutation, pre-ligand-binding relaxation of the backbone dihedral angles at position 137 in the tyrosine gate and their coupling to Tyr48 via the interiorly located Ile52 form the basis of the loss of affinity of the FimH adhesin in the Y137A mutant

Cadmium phytotoxicity: issues, progress, environmental concerns and future perspectives

Cadmium, a high toxicity element, is a potential threat to plant and human health, and a dangerous pollutant in the environment. Uptake and accumulation by crops represent the main entry pathway for potentially health-threatening toxic metals into human and animal food. Crops and other plants take up Cd from the soil or water and may distribute it in their roots and shoots. Soil and/or water are usually contaminated with Cd through natural sources, industrial effluent, and anthropogenic activities. In this review, the sources of Cd contamination, evaluation of the phytotoxic effects on plants, and mode of action of Cd toxicity, were summarized. Plant defensive strategies upon excess Cd are also considered in this review. Cd-induced effects include oxidative stress, disintegration of the photosynthetic apparatus, reduction in gas exchange parameters, nutrient imbalance, and subcellular organelle degradation. In addition, Cd severely impairs biomolecules such as DNA, protein, and lipids. Although plants are sessile in nature, they are equipped with certain mechanisms to cope with unfavorable conditions. These mechanisms include synthesis of metal-helating proteins, expression of enzymatic and non-enzymatic antioxidants, organic acids, and plant root–mycorrhiza association. The built-in system of plant tolerance to Cd can be further enhanced by the application of exogenous organic and inorganic metal sources. This review will broaden the knowledge about the Cd accumulation in plants and the responses to metal exposure, as well as our understanding of metal tolerance and overcoming this serious issue for sustainable agriculture and human health worldwide. Highlights Cd accumulation has harmful effects in an organism. Cd has been listed 7th out of 275 compounds in the priority list of hazardous materials. Cd remains in the soil for 15–1100 years. Plants usually imply certain strategies to overcome Cd toxicity. Plants built-in systems can be enhanced to overwhelmed this problem.Cadmium, a high toxicity element, is a potential threat to plant and human health, and a dangerous pollutant in the environment. Uptake and accumulation by crops represent the main entry pathway for potentially health-threatening toxic metals into human and animal food. Crops and other plants take up Cd from the soil or water and may distribute it in their roots and shoots. Soil and/or water are usually contaminated with Cd through natural sources, industrial effluent, and anthropogenic activities. In this review, the sources of Cd contamination, evaluation of the phytotoxic effects on plants, and mode of action of Cd toxicity, were summarized. Plant defensive strategies upon excess Cd are also considered in this review. Cd-induced effects include oxidative stress, disintegration of the photosynthetic apparatus, reduction in gas exchange parameters, nutrient imbalance, and subcellular organelle degradation. In addition, Cd severely impairs biomolecules such as DNA, protein, and lipids. Although plants are sessile in nature, they are equipped with certain mechanisms to cope with unfavorable conditions. These mechanisms include synthesis of metal-helating proteins, expression of enzymatic and non-enzymatic antioxidants, organic acids, and plant root–mycorrhiza association. The built-in system of plant tolerance to Cd can be further enhanced by the application of exogenous organic and inorganic metal sources. This review will broaden the knowledge about the Cd accumulation in plants and the responses to metal exposure, as well as our understanding of metal tolerance and overcoming this serious issue for sustainable agriculture and human health worldwide. Highlights Cd accumulation has harmful effects in an organism. Cd has been listed 7th out of 275 compounds in the priority list of hazardous materials. Cd remains in the soil for 15–1100 years. Plants usually imply certain strategies to overcome Cd toxicity. Plants built-in systems can be enhanced to overwhelmed this problem

Tetra- and Hexavalent Siglec-8 Ligands Modulate Immune Cell Activation

Carbohydrate-binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent-exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec-8, a monomeric I-type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra- and hexavalent and, to an even larger extent, multivalent ligands to Siglec-8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect