Caspase-1 activation by Salmonella

Salmonella is an interesting example of how the selective pressure of host environments has led to the evolution of sophisticated bacterial virulence mechanisms. This microbe exploits the first-line of defence, the macrophage, as a crucial tool in the initiation of disease. After invasion of intestinal macrophages, a virulence protein secreted by Salmonella specifically induces apoptotic cell death by activating the cysteine protease caspase-1. The pro-apoptotic capability is necessary for successful pathogenesis. The study of mechanisms by which Salmonella induces programmed cell death offers new insights into how pathogens cause disease and into general mechanisms of activation of the innate immune system.     

More is not necessarily better: prozone-like effects in passive immunization with IgG

Despite a century of study, the relationship between Ag-specific Ig concentration and protection remains poorly understood for the majority of pathogens. In certain conditions, administration of high Ab doses before challenge with an infectious agent can be less effective than smaller Ab doses, a phenomenon which is consistent with a prozone-like effect. In this study, the relationship between IgG1, IgG2a, IgG2b, and IgG3 dose, infective inocula, and protection was investigated in a mouse model of Cryptococcus neoformans infection. The activity of each IgG subclass ranged from protective to disease-enhancing depending on both the Ab dose and infective inocula used. Enhanced dissemination to the brain was observed in mice given a high IgG2a dose and a relatively low inoculum. Ab administration had immunomodulatory effects, with cytokine expression in lung, brain, and spleen varying as a function of the infective inoculum Ab dose and IgG subclass. In vitro studies did not predict or explain the mechanism of in vivo prozone-like effects, because all isotypes were opsonic and elicited NO release from macrophages. IgG2a was most efficient in inducing a macrophage oxidative burst. These results reveal that an individual Ab can be protective, nonprotective, or disease-enhancing depending on its concentration relative to a challenge inoculum. Our findings have implications for the potential contribution of Ab responses to defense against microbial diseases because Ab-mediated immunity may be protective, nonprotective, or even deleterious to the host.     

A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8(+) T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8(+) T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8(+) T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.     

In vitro protein-DNA interactions at the human lamin B2 replication origin

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition.     

Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme

Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.     

Feeding habits of guanacos<Emphasis Type="Italic">Lama guanicoe</Emphasis> in the high Andes of north-central Chile

The guanacoLama guanicoe Muller, 1776 has a wide distribution along the Andes and Patagonia. We studied the feeding behaviour of a guanaco population that lives over 4100 m altitude in the Andes of north-central Chile. By contrasting the diet of guanacos during a dry year with that of a wet year and comparing it with the plant availabilities in the field, we tested the hypothesis that the guanaco is a generalist herbivore. We predicted that under such extreme habitat conditions guanacos should consume whatever plant species are available in the environment, especially in a dry year, when vegetation is scarcer. In addition, we compared its diet at three different age classes. We estimated the diet through the microhistological analysis of plant remains found in guanaco pellets collected during January of 1997 (ie after a dry year) and 1998 (ie after a wet year; 41 vs 495 mm, respectively). Then, we computed dietary preferences, food niche-breadth, and food-niche overlap between years and among age classes. Vegetation cover and plant species richness were higher during the wet than during the dry year. The most common plants in the environment were perennial graminoids and legumes. Contrary to our prediction, the guanaco preferred a few plant species, showing a relatively narrow diet breadth that changed little between years differing in plant abundances. The diet proportions differed among the three age classes, however. Our data indicate that at least in this high-elevation population, guanacos are selective and non-opportunistic herbivores. This specialized feeding behaviour is puzzling given the energetic demands of living in a harsh environment with low availabilities of resources. The hypothesis that this is due to the lower palatability of the plants not eaten, remains to be tested.     

Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation

Fermentation conditions and microorganisms were determined, based on acid production, glucose concentration as carbohydrate source. Inoculation levels to obtain a stable shrimp waste silage were also determined. Shrimp waste ensilation was an efficient method of preservation, allowing the recovery of chitin and another added-value products such as pigments, proteins and enzymes. From the various lactic acid bacteria tested, Lactobacillus pentosus and Lactobacillus sp. (B2) were the best lactic acid producers, although small quantities of acetic acid were detected in samples inoculated with Lactobacillus pentosus. Therefore B2 was chosen for the analysis of glucose consumption as well as for the determination of optimum inoculation levels. The best results were obtained at 10% (w/w wet basis) and 5% (v/w wet basis) respectively. Presence of starters and initial glucose concentration were critical factors in the fermentation of shrimp waste. High initial glucose and starter concentrations reduced the time and increased the amount of lactic acid produced. The fermentation pattern changed during ensilation from hetero to homofermentative. Shrimp waste ensilation prevented the growth of spoilage microorganisms keeping their microbial counts steady and pH values within the acid region.