Adsorption of human serum proteins onto TREN-agarose: Purification of human IgG by negative chromatography

Tris(2-aminoethyl)amine (TREN) – a chelating agent used in IMAC – immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90–95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.     

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5' extremity of each mRNA, supplying the 5'-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania (Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.     

XRCC1 gene polymorphisms in a population sample and in women with a family history of breast cancer from Rio de Janeiro (Brazil)

The X-ray repair cross-complementing Group1 (XRCC1) gene has been defined as essential in the base excision repair (BER) and single-strand break repair processes. This gene is highly polymorphic, and the most extensively studied genetic changes are in exon 6 (Arg194Trp) and in exon 10 (Arg399Gln). These changes, in conserved protein sites, may alter the base excision repair capacity, increasing the susceptibility to adverse health conditions, including cancer. In the present study, we estimated the frequencies of the XRCC1 gene polymorphisms Arg194Trp and Arg399Gln in healthy individuals and also in women at risk of breast cancer due to family history from Rio de Janeiro. The common genotypes in both positions (194 and 399) were the most frequent in this Brazilian sample. Although the 194Trp variant was overrepresented in women reporting familial cases of breast cancer, no statistically significant differences concerning genotype distribution or intragenic interactions were found between this group and the controls. Thus, in the population analyzed by us, variants Arg194Trp and Arg399Gln did not appear to have any impact on breast cancer susceptibility.     

Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man₅GlcNAc₂ to Man₉GlcNAc₂ glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37°C, it is essential for parasite growth and survival at 40°C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.Trypanosoma brucei is a protozoan parasite with two main proliferative stages in its life cycle: the procyclic form that grows in the tsetse fly midgut, and the bloodstream form that causes African sleeping sickness in humans and nagana in cattle. The bloodstream form is covered in a densely packed layer of 5 × 10⁶ glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) dimers. This coat protects the parasites from the alternative pathway of complement-mediated lysis, shields other cell surface proteins from the host immune system, and by the process of antigenic variation allows these parasites to persist for long periods in the host bloodstream (16, 54). The trypanosome genome contains several hundreds of silent VSG genes, most of which are pseudogenes in subtelomeric arrays (40). T. brucei evades host-acquired immunity through differential activation of these genes, which encode immunologically distinct GPI-anchored glycoproteins with one to three N glycosylation sites (27, 43).Protein N glycosylation is the most common covalent protein modification in eukaryotic cells (25). N-glycans contribute to “quality control” in the endoplasmic reticulum (ER) through a series of oligosaccharide-processing and lectin-binding reactions that contribute to protein folding and the targeting of misfolded glycoproteins for degradation (24, 47, 58, 65). As nascent protein chains enter the ER lumen, they are modified covalently in most eukaryotes by the addition of the Glc₃Man₉GlcNAc₂ core glycan via the action of oligosaccharyltransferase (OST). After deglucosylation by α-glucosidases I (GI) and II (GII), misfolded glycoproteins can be reglucosylated in the ER by the UDP-Glc:glycoprotein glucosyltransferase (UGGT), recreating the same monoglucosylated trimming intermediate generated by GII (9, 64, 66). UGGT behaves as a sensor of glycoprotein conformation and is a key constituent of ER quality control (50, 61). Calnexin and calreticulin are ER-resident lectin-like quality control chaperones that recognize the monoglucosylated glycans on glycoproteins and help them to fold properly through their close association with the oxidoreductase ERp57 (49). On reaching the proper tertiary structure, the glycoproteins are still substrates of GII but no longer of UGGT. Properly folded molecules, thus liberated from the lectins, are then free to continue their transit to the Golgi apparatus (64). When exposure to the folding machinery in the ER is not sufficient to promote a native conformation, proteins are eventually degraded by ER-associated degradation (49, 64).Most eukaryotes under conditions of stress, such as heat shock, undergo an unfolded protein response (UPR) that is triggered by sensing unfolded proteins in the ER. The UPR typically leads to increased expression of ER quality control components, such as calnexin and calreticulin and the ER chaperone Gpr78/BiP, as well inhibition of protein synthesis and cell cycle arrest (53, 57, 60).In contrast to the situation in most other eukaryotes, none of the trypanosomatid dolichol-linked oligosaccharides are capped with glucose residues, as these parasites do not synthesize the sugar donor dolichol-phosphate-glucose for these reactions (41, 59). The mature dolichol-phosphate-oligosaccharide species used for transfer to protein vary according to trypanosomatid species (17, 51, 52, 56). Therefore, in these organisms, monoglucosylated glycans are exclusively formed through UGGT-dependent glucosylation (12). Furthermore, trypanosomatids lack calnexin, which binds and participates in the refolding of glucosylated proteins, and it has been suggested that differences in the N-glycan precursor have profound effects on N-glycan-dependent quality control of glycoprotein folding and ER-associated degradation (4). These protozoa do not present a conventional OST complex and express only the catalytic stt3 protein subunit that, at least for the Trypanosoma cruzi and Leishmania major enzymes, shows little specificity toward the structure of the dolichol-phosphate-oligosaccharide donor (4, 11, 26, 31, 32, 45). In the case of T. brucei, while the insect-dwelling procyclic form makes and transfers Man₉GlcNAc₂-phosphate-dolichol (1), previous work from our group showed that the bloodstream form of the parasite transfers both Man₉GlcNAc₂ and Man₅GlcNAc₂ to VSG in a site-specific manner (29). Regarding ER folding and quality control, although in vitro assays have shown that T. cruzi and higher eukaryotic UGGTs exclusively glucosylate high-mannose glycans in misfolded glycoproteins (66), in T. brucei the UGGT and GII enzymes use Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂, respectively, as their substrates in the processing of VSG variant 221 (VSG221) (29). However, it could not be concluded from that study whether this apparent preference for atypical biantennary Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂ structures reflected the substrate specificity of the enzymes or the location of the glycosylation site in the VSG polypeptide chain (30).In this work, we further analyze the specificity and function of the UGGT/GII quality control system of T. brucei by analyzing the non-VSG N-glycans of our α-GII null mutant and creating and characterizing a T. brucei UGGT null mutant.     

Synthesis, tuberculosis inhibitory activity, and SAR study of N-substituted-phenyl-1,2,3-triazole derivatives

The aim of this work was to describe the synthesis, the in vitro anti-Mycobacterium tuberculosis profile, and the structure–activity relationship (SAR) study of new N-substituted-phenyl-1,2,3-triazole-4-carbaldehydes (3a–l). The reactions of aromatic amine hydrochlorides with diazomalonaldehyde (1) produced several N-substituted-phenyl-1,2,3-triazole-4-carbaldehydes (3a–l) in moderate-to-good yields. In order to investigate the influence of the difluoromethylene group on the anti-Mycobacterium activity of these compounds, fluorination of triazoles with DAST converted the corresponding carbaldehyde compounds into new difluoromethyl derivatives (4a–l) in excellent yield. Characterization of all compounds was achieved by spectroscopic means and additional for 1-(4-methylphenyl)-1,2,3-triazole-4-carbaldehyde, 3k by X-ray crystallography. Compounds (3a–l) and (4a–l) have been screened for the inhibitory activity against Mycobacterium tuberculosis H37Rv strain (ATCC 27294) and all of them were able to inhibit the growth of the bacterium. Interestingly, 3a and 3k exhibited the best inhibition with MIC values of 2.5 μg/mL, similar to pharmaceuticals currently used in the treatment of tuberculosis. Our SAR study indicated the importance of the hydrogen bond acceptor subunit (3a–l), the position in the aromatic ring, the planarity of triazole and phenyl rings in these compounds, and a correlation between the uniform HOMO coefficient distribution and the anti-tubercular activity. The significant activity of 3a and 3k pointed them as promising lead molecules for further synthetic and biological exploration.     

Bacterial Community Associated with Healthy and Diseased Reef Coral Mussismilia hispida from Eastern Brazil

In order to characterize the bacterial community diversity associated to mucus of the coral Mussismilia hispida, four 16S rDNA libraries were constructed and 400 clones from each library were analyzed from two healthy colonies, one diseased colony and the surrounding water. Nine bacterial phyla were identified in healthy M. hispida, with a dominance of Proteobacteria, Actinobacteria, Acidobacteria, Lentisphaerae, and Nitrospira. The most commonly found species were related to the genera Azospirillum, Hirschia, Fabibacter, Blastochloris, Stella, Vibrio, Flavobacterium, Ochrobactrum, Terasakiella, Alkalibacter, Staphylococcus, Azospirillum, Propionibacterium, Arcobacter, and Paenibacillus. In contrast, diseased M. hispida had a predominance of one single species of Bacteroidetes, corresponding to more than 70% of the sequences. Rarefaction curves using evolutionary distance of 1% showed a greater decrease in bacterial diversity in the diseased M. hispida, with a reduction of almost 85% in OTUs in comparison to healthy colonies. ∫-Libshuff analyses show that significant p values obtained were <0.0001, demonstrating that the four libraries are significantly different. Furthermore, the sympatric corals M. hispida and Mussismilia braziliensis appear to have different bacterial community compositions according to Principal Component Analysis and Lineage-specific Analysis. Moreover, lineages that contribute to those differences were identified as α-Proteobacteria, Bacteroidetes, and Firmicutes. The results obtained in this study suggest host–microbe co-evolution in Mussismilia, and it was the first study on the diversity of the microbiota of the endemic and endangered of extinction Brazilian coral M. hispida from Abrolhos bank.     

Seasonal functional relevance of sperm characteristics in equine spermatozoa

A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and lastly by fertility, stressing the importance of individual variations in out-bred animal models. The analysis of multiple ejaculates from the same animals showed clear seasonal-based differences (P < 0.05) with poor semen quality in winter and a noticeable improvement in sperm quality with increasing photoperiod. Better semen quality was observed between late March and May. Interactions between month period, animal, and fertility were evident (P < 0.05) for sperm concentration, head and tail sperm anomalies, and acrosomal integrity. Thus, it may be advisable to adjust the use of stallion semen according to seasonal variations.     

Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4% and 78.7%, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.