Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.     

A geometric and algebraic view of MHC-peptide complexes and their binding properties

Background  

Major histocompatibility complex (MHC) molecules present peptides to T lymphocytes. It is of critical biological and medical importance to elucidate how different MHC alleles bind to a specific set of peptides.     

Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation

INTRODUCTIONThe inappropriate enhancement of lymphocytesurviVal due to a block in programmed cell deathand/or an enhancement of entry into the cell cyclecan contribute to the abnormal expansion of clonesresulting in tumorigenesis or the breakdown of pe-ripheral self-toleranced, 2]. Proper lymphocytehomeostasis is critical for normal immune functionand is maintained by a complex series of cellularinteractions and the action of secreted cytokines.Illterleukin-4 (IL-4), a cytokine produced …     

Mutations in foregut SOX2+ cells induce efficient proliferation via CXCR2 pathway

Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2⁺ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as Kras^G12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2⁺ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and Kras^G12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.     

Changes in mating behavior, erectile function, and nitric oxide levels in penile corpora cavernosa in streptozotocin-diabetic rats.

This study assessed whether the in vivo production of nitric oxide (NO) in the penis is impaired in experimental diabetes and whether this phenomenon can be explained by abnormal levels of NO synthase isoenzymes and/or plasma androgens. Adult male Sprague-Dawley rats were injected with streptozotocin (STZ) (40 mg/kg, i.p.) or vehicle. One half of the STZ-treated animals received daily insulin replacement. Twelve weeks later, the animals were tested for mating behavior and erectile reflexes. They were then anesthetized with urethane (1 g/kg), and the NO levels in their corpora cavernosa were monitored electrochemically with porphyrin microsensors before and after electrostimulation of the cavernous nerve. The intracavernous pressure (ICP) was measured simultaneously. The diabetic animals had substantial impairment in the mating and erectile reflexes tests, decreased basal and stimulated NO levels in the corpora, and a reduced ICP response to cavernous nerve stimulation. Insulin replacement fully reversed the effects of diabetes on the mating reflexes, the basal NO signals, and the ICP responses to electrical field stimulation and partially restored the stimulated NO release. Neither diabetes nor diabetes with insulin treatment had significant effects on serum testosterone levels or NOS isoform (nNOS, eNOS, and iNOS) protein content in penile homogenates, indicating that the changes found in erectile function were independent of such variables. These results also suggest that the diabetes-induced reduction in corporeal NO levels could be mainly due to the lack of some essential cofactors for NOS activity rather than to changes in the amount of enzyme proteins.     

Averrhoidium dalyi (Sapindaceae): a new species from western Amazonia

The present paper describes and illustrates the new speciesAverrhoidium dalyi and provides a key to the species ofAverrhoidium. The new species is known from lowland, terra firme forest in the Purus River basin, in Acre, Brazil, and from Manu National Park in Madre de Dios, Peru.

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Resumen  En el presente artículo se describe, discute e ilustra la nueva especieAverrhoidium dalyi coleccionada en la cuenca del Río Purus en el estado de Acre, Brasil y en el Parque Nacional del Manu en el departamento Madre de Dios, Peru. También en el presente se provee una clave para las especies deAverrhoidium.

     

Two new species of Serjania (Sapindaceae) from southeastern Brazil

One new species ofSerjania (S. erythrocaulis) belonging to theS. paradoxa group ofSerjania sect.Eurycoccus is described and illustrated bringing the total number of species in this group to eight. A second new species,Serjania divaricocca, belonging to sect.Serjania is also described and illustrated.

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Resumen  Una especie neuva deSerjania (S. erythrocaulis) perteneciente al grupoS. paradoxa de la secciónEurycoccus es descrita e ilustrada. Con esta especie, el numero total de especies conocidas para este grupo aumenta a ocho. Una segunda nueva especie,Serjania divaricocca, perteneciente a la secciónSerjania es también descrita e ilustrada.

     

Cellulase isoenzyme profiles in Frankia strains belonging to different cross-inoculation groups

Carboxymethyl cellulase activities were evaluated in eight strains of Frankia from diverse host specificity groups and geographical origins. Cellulase activity was detected in culture supernatants of all strains in the absence of CMC (Carboxymethylcellulose) as an inducer using both double-layer plate and reducing sugar assays, indicating a constitutive production of CM-cellulases by Frankia. CM-cellulase isoenzyme profiles were visualized using activity gel electrophoresis of concentrated culture supernatants. Different electrophoretic profiles were observed among the eight strains tested, which correlate with the host specificity and taxonomic grouping of Frankia.     

Characterization of Rhizobial Isolates of Phaseolus vulgaris by Staircase Electrophoresis of Low-Molecular-Weight RNA

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.     

Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli

Submergence stimulates elongation of the leaves of Rumex palustris and under laboratory conditions the maximum final leaf length (of plants up to 7 weeks old) was obtained within a 9 day period. This elongation response, mainly determined by petiole elongation, depends on the availability of storage compounds and developmental stage of a leaf. A starch accumulating tap root and mature leaves and petioles were found to supply elongating leaves with substrates for polysaccharide synthesis in expanding cell walls. Changes in the composition of cell wall polysaccharides of elongated petioles suggest a substantial cell wall metabolism during cell extension. Reduced starch levels or removal of mature leaves caused a substantial limitation of submerged leaf growth. From the 5th leaf onward enough reserves were available to perform submerged leaf growth from early developmental stages. Very young petioles had a limited capacity to elongate. In slightly older petioles submergence resulted in the longest final leaf lengths and these values gradually decreased when submergence was started at more mature developmental stages. Submerged leaf growth is mainly a matter of petiole elongation in which cell elongation has a concurrent synthesis of xylem elements in the vascular tissue. Mature petioles still elongated (when submerged) by cell and tissue elongation only: the annular tracheary elements stretched enabling up to 70% petiole elongation.