RESUMO
Glycans, carbohydrate molecules in the realm of biology, are present as biomedically important glycoconjugates and a characteristic aspect is that their structures in many instances are branched. In determining the primary structure of a glycan, the sugar components including the absolute configuration and ring form, anomeric configuration, linkage(s), sequence, and substituents should be elucidated. Solution state NMR spectroscopy offers a unique opportunity to resolve all these aspects at atomic resolution. During the last two decades, advancement of both NMR experiments and spectrometer hardware have made it possible to unravel carbohydrate structure more efficiently. These developments applicable to glycans include, inter alia, NMR experiments that reduce spectral overlap, use selective excitations, record tilted projections of multidimensional spectra, acquire spectra by multiple receivers, utilize polarization by fast-pulsing techniques, concatenate pulse-sequence modules to acquire several spectra in a single measurement, acquire pure shift correlated spectra devoid of scalar couplings, employ stable isotope labeling to efficiently obtain homo- and/or heteronuclear correlations, as well as those that rely on dipolar cross-correlated interactions for sequential information. Refined computer programs for NMR spin simulation and chemical shift prediction aid the structural elucidation of glycans, which are notorious for their limited spectral dispersion. Hardware developments include cryogenically cold probes and dynamic nuclear polarization techniques, both resulting in enhanced sensitivity as well as ultrahigh field NMR spectrometers with a 1H NMR resonance frequency higher than 1 GHz, thus improving resolution of resonances. Taken together, the developments have made and will in the future make it possible to elucidate carbohydrate structure in great detail, thereby forming the basis for understanding of how glycans interact with other molecules.
RESUMO
Sclerosing encapsulating peritonitis (SEP) is a rare condition which consists of reactive fibrous tissue proliferation with mixed inflammatory infiltration within the abdominal cavity. The present report describes an additional case of SEP affecting a mixed-breed immature female dog presented with persistent vomiting, progressive weight loss, and ascites. Abdominal radiographic and ultrasonographic findings suggested abdominal neoformation resulting in gastric displacement, in addition ascitic fluid was evaluated and cytology showed large numbers of inflammatory effusion. An exploratory laparotomy was performed and were detected multiple thick peritoneal adhesions which restricted mobility of abdominal viscera. Biopsy specimens of these lesions were submitted for histopathological examination. Microscopically, the external serous surfaces of the abdominal organs were covered with dense fibrous connective tissue characterized by intense mature collagen deposition and moderate angiogenesis. The animal was euthanized due to poor prognosis and sent for necropsy. The clinical, imaging, gross and microscopic findings were compatible with SEP.(AU)
Assuntos
Animais , Peritonite/diagnóstico por imagem , Doenças do Cão , Cães , Obstrução Intestinal/diagnóstico por imagem , Laparotomia/métodosRESUMO
An immunoadjuvant preparation (named Fraction B) was obtained from the aqueous extract of Quillaja brasiliensis leaves, and further fractionated by consecutive separations with silica flash MPLC and reverse phase HPLC. Two compounds were isolated, and their structures elucidated using a combination of NMR spectroscopy and mass spectrometry. One of these compounds is a previously undescribed triterpene saponin (Qb1), which is an isomer of QS-21, the unique adjuvant saponin employed in human vaccines. The other compound is a triterpene saponin previously isolated from Quillaja saponaria bark, known as S13. The structure of Qb1 consists of a quillaic acid residue substituted with a ß-d-Galp-(1â2)-[ß-d-Xylp-(1â3)]-ß-d-GlcpA trisaccharide at C3, and a ß-d-Xylp-(1â4)-α-l-Rhap-(1â2)-[α-l-Arap-(1â3)]-ß-d-Fucp moiety at C28. The oligosaccharide at C28 was further substituted at O4 of the fucosyl residue with an acyl group capped with a ß-d-Xylp residue.
Assuntos
Saponinas , Triterpenos , Adjuvantes Imunológicos/química , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Quillaja/química , Saponinas/química , Triterpenos/químicaRESUMO
Canine morbillivirus, also known as canine distemper virus (CDV), induces a contagious multisystemic disease caused by an enveloped RNA virus belonging to the genus Morbillivirus within the family Paramyxoviridae. CDV replicates readily in epithelial, nerve and lymphoid tissues; it is excreted in urine, feces, saliva, oral and nasal discharge; and its major route of entry for infection is through the respiratory system. Although the virus was originally believed to infect domestic dogs, new studies have shown that it can also naturally or experimentally infect non-domestic hosts. A recent blood test performed on a giant anteater (Myrmecophaga tridactyla) found Lentz inclusions in the animal's leucocytes. A rapid CDV test, an RT-PCR assay and pathology findings confirmed this report of canine morbillivirus in this species, which corresponds to the second report of CDV infection in the order Pilosa, family Myrmecophagidae in central west Brazil.
Assuntos
Vírus da Cinomose Canina/isolamento & purificação , Cinomose/virologia , Eutérios , Animais , Animais de Zoológico , BrasilRESUMO
Bisthiazolidines (BTZ) are bicyclic compounds considered penicillin analogs that inhibit the full range of Metallo-ß-Lactamases (MBLs) and potentiate ß-lactam activity against resistant bacteria. Herein we present a new methodology to prepare 2-substituted bisthiazolidines by aldehyde exchange. Thirteen new bisthiazolidines were prepared using this methodology, with yields ranged from 31 to 75%. The reaction is based on in situ imines formation, which are able to exchange side chains. The reaction intermediates were studied based on NMR experiments and a key imine 1b-II could be detected in the reaction mixture. Furthermore, a DFT computational analysis was performed to gain insights into the reaction mechanism, allowing us to unveil the different pathways and their activation barriers within the synthetic route. The results suggest that the most favorable route involve the formation of the thiazolidine 1b-III by i) a N-assisted N-C bond cleavage, and ii) a thiol-mediated 5 endo-trig cyclization followed by a C-N bond cleavage. In contrast with previously reported evidence, the imine metathesis was discarded as a plausible pathway. Finally, the reaction of 1b-III with aldehyde 2a leads to bicycle 4a via the iminium ion 1b-V.
RESUMO
O objetivo do presente relato foi avaliar a viabilidade da técnica de coleta de células espermáticas do epidídimo de um cervo dama (Dama dama) após orquiectomia e comparar sua capacidade de criopreservação utilizando dois diluidores comerciais diferentes, o Triladyl® e o Andromed®. Foram avaliados o volume testicular, a motilidade e vigor espermáticos e a morfologia das células espermáticas antes e após o processo de criopreservação. Ao se comparar os dois meios comerciais, pode-se constatar que a motilidade e vigor espermáticos foram melhores na alíquota congelada com Triladyl® (25% e 2) do que com Andromed® (10% e 1). Quanto a morfologia espermática os dois diluidores apresentaram valores semelhantes de defeitos totais (32.5% com Andromed® e 33% com Triladyl®, sendo a cabeça lanciforme o defeito predominante em ambos os casos). Diante desses resultados, a técnica de coleta de células espermáticas do epidídimo é viável para cervídeos Dama e mais estudos são necessários para se estabelecer o melhor meio diluidor para esta espécie criada em climas tropicais.
The aim of the present report was to evaluate the viability of epididymal sperm collection technique in a fallow deer (Dama dama) after orchiectomy and to compare its cryopreservation capacity using two different commercial extenders, Triladyl® and Andromed®. Thus, testicular volume, sperm motility and vigor, and sperm cell morphology were evaluated before and after the cryopreservation process. When comparing the two commercial media, sperm motility and vigor were better in the post-thawed aliquot extended with Triladyl® (25% and 2) than with Andromed® (10% and 1). Regarding sperm morphology, the two dilutors presented similar values of total defects (32.5% with Andromed® and 33% with Triladyl®, with the lanciform head being the predominant defect in both cases). Given these results, the epididymal sperm cell collection technique is viable for Dama cervids and further studies are needed to establish the best extender for this species raised under tropical conditions.
Assuntos
Masculino , Animais , Cervos/anatomia & histologia , Criopreservação/veterinária , Orquiectomia/veterinária , EpididimoRESUMO
O objetivo do presente relato foi avaliar a viabilidade da técnica de coleta de células espermáticas do epidídimo de um cervo dama (Dama dama) após orquiectomia e comparar sua capacidade de criopreservação utilizando dois diluidores comerciais diferentes, o Triladyl® e o Andromed®. Foram avaliados o volume testicular, a motilidade e vigor espermáticos e a morfologia das células espermáticas antes e após o processo de criopreservação. Ao se comparar os dois meios comerciais, pode-se constatar que a motilidade e vigor espermáticos foram melhores na alíquota congelada com Triladyl® (25% e 2) do que com Andromed® (10% e 1). Quanto a morfologia espermática os dois diluidores apresentaram valores semelhantes de defeitos totais (32.5% com Andromed® e 33% com Triladyl®, sendo a cabeça lanciforme o defeito predominante em ambos os casos). Diante desses resultados, a técnica de coleta de células espermáticas do epidídimo é viável para cervídeos Dama e mais estudos são necessários para se estabelecer o melhor meio diluidor para esta espécie criada em climas tropicais.(AU)
The aim of the present report was to evaluate the viability of epididymal sperm collection technique in a fallow deer (Dama dama) after orchiectomy and to compare its cryopreservation capacity using two different commercial extenders, Triladyl® and Andromed®. Thus, testicular volume, sperm motility and vigor, and sperm cell morphology were evaluated before and after the cryopreservation process. When comparing the two commercial media, sperm motility and vigor were better in the post-thawed aliquot extended with Triladyl® (25% and 2) than with Andromed® (10% and 1). Regarding sperm morphology, the two dilutors presented similar values of total defects (32.5% with Andromed® and 33% with Triladyl®, with the lanciform head being the predominant defect in both cases). Given these results, the epididymal sperm cell collection technique is viable for Dama cervids and further studies are needed to establish the best extender for this species raised under tropical conditions.(AU)
Assuntos
Animais , Masculino , Cervos/anatomia & histologia , Criopreservação/veterinária , Orquiectomia/veterinária , EpididimoRESUMO
Searching for prospective vanadium-based agents against Trypanosoma cruzi, the parasite causing Chagas disease, four new [VVO(8HQ-H)(L-2H)] compounds, where 8HQ is 8-hydroxyquinoline and L are tridentate salicylaldehyde semicarbazone derivatives L1-L4, were synthesized and characterized in the solid state and in solution. The compounds were evaluated on T. cruzi epimastigotes (CL Brener) as well as on VERO cells, as mammalian cell model. Compounds showed activity against T. cruzi (IC50 6.2-10.5 µM) of the same order than Nifurtimox and 8HQ, and a four- to sevenfold activity increase with respect to the free semicarbazones. For comparison, [VVO2(L-H)] series was prepared and the new [VVO2(L3-H)] was fully characterized. They showed negligible activity and low selectivity towards the parasite. The inclusion of 8HQ as ligand in [VVO(8HQ-H)(L-2H)] compounds led to good activities and increased selectivity towards the parasite with respect to 8HQ. 51V NMR experiments, performed to get insight into the nature of the active species, suggested partial decomposition of the compounds in solution to [VVO2(L-H)] and 8HQ. Depending on the dose, the compounds act as trypanocide or trypanostatic. A high uptake of vanadium in the parasites (58.51-88.9% depending on dose) and a preferential accumulation in the soluble protein fraction of the parasite was determined. Treated parasites do not seem to show a late apoptotic/necrotic phenotype suggesting a different cell death mechanism. In vivo toxicity study on zebrafish model showed no toxicity up to a 25 µM concentration of [VVO(8HQ-H)(L1-2H)]. These compounds could be considered prospective anti-T. cruzi agents that deserve further research.
Assuntos
Complexos de Coordenação/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Vanádio/química , Animais , Apoptose/efeitos dos fármacos , Chlorocebus aethiops , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Testes de Sensibilidade Parasitária , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/toxicidade , Células Vero , Peixe-ZebraRESUMO
The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpß1-3 residue can initiate a chain comprising one to three additional Galpß1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpß1-6 residues. These, as well as the GlcNAcpß1-6 residue in core 2, can be decorated with the Galpα1-4Galpß1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpß1-3(Galpα1-4Galpß1-4GlcNAcpß1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpß1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc ß1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis.
Assuntos
Echinococcus granulosus/química , Echinococcus multilocularis/química , Mucinas/química , Polissacarídeos/química , Animais , Cromatografia em Gel , Mineração de Dados , Echinococcus granulosus/classificação , Echinococcus granulosus/genética , Echinococcus multilocularis/classificação , Echinococcus multilocularis/genética , Regulação Enzimológica da Expressão Gênica , Genoma , Gerbillinae , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Larva/química , Larva/classificação , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Mucinas/metabolismo , Cavidade Peritoneal/parasitologia , Polissacarídeos/biossíntese , Polissacarídeos/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
We present a detailed conformational study of a simplified synthetic analog of the bis-oxazole oxane fragment found in the cytostatic agents phorboxazole A and B based on results from NMR spectroscopy and molecular modeling simulations. Complete 1H and 13C resonance assignments for the bis-oxazole oxane system were carried out through the use of COSY, HSQC, HMBC, TOCSY, and HSQC-TOCSY experiments, and its conformational preferences in solution were investigated by analysis of 3J(HH) coupling constants and NOE enhancements obtained from 1D and 2D NOESY experiments. In order to solve inconsistencies from our preliminary structural studies, simulated annealing studies were performed to thoroughly sample the phase space available to the molecule. Our results reveal that the six-membered oxane ring, which constitutes the most important moiety regarding the three-dimensional (3D) structure and flexibility of the analog, exists in rapid equilibrium between its two accessible chair conformers in an approximate ratio of 70:30. The information gathered from these studies will be of critical importance in our efforts to prepare novel compounds with phorboxazole-like structure and activity.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/química , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Oxazóis/química , Conformação MolecularRESUMO
The abundant metabolite myo-inositol hexakisphosphate (InsP6) can form vesicular deposits with cations, a widespread phenomenon in plants also found in the cestode parasite, Echinococcus granulosus. In this organism, the deposits are exocytosed, accumulating in a host-exposed sheath of extracellular matrix termed the laminated layer. The formation and mobilization of InsP6 deposits, which involve precipitation and solubilization reactions, respectively, cannot yet be rationalized in quantitative chemical terms, as the solids involved have not been formally described. We report such a description for the InsP6 deposits from E. granulosus, purified as the solid residue left by mild alkaline digestion of the principal mucin component of the laminated layer. The deposits are largely composed of the compound Ca5H2L.16H2O (L representing fully deprotonated InsP6), and additionally contain Mg2+ (6-9% molar ratio with respect to Ca2+), but not K+. Calculations employing recently available chemical constants show that the precipitation of Ca5H2L.16H2O is predicted by thermodynamics in secretory vesicle-like conditions. The deposits appear to be similar to microcrystalline solids when analysed under the electron microscope; we estimate that each crystal comprises around 200 InsP6 molecules. We calculate that the deposits increase, by three orders of magnitude, the surface area available for adsorption of host proteins, a salient ability of the laminated layer. The major inositol phosphate in the deposits, other than InsP6, is myo-inositol (1,2,4,5,6) pentakisphosphate, or its enantiomer, inositol (2,3,4,5,6) pentakisphosphate. The compound appears to be a subproduct of the intracellular pathways leading to the synthesis and vesicular accumulation of InsP6, rather than arising from extracellular hydrolysis of InsP6.