Effects of free ATP, citrate, and bicarbonate on rat liver mitochondrial ATPase.

The effects of citrate, free ATP, and bicarbonate on the activity of isolated rat liver mitochondrial ATPase have been studied. Citrate behaved as a weak noncompetitive inhibitor; free ATP at low concentrations was found to be an activator, whereas at high concentrations behaved as an inhibitor. Citrate competed with free ATP. Neither citrate nor free ATP competed with bicarbonate. The results obtained suggest the existence of at least two catalytic sites with ATP hydrolyzing activity and at least three regulatory sites: one for bicarbonate, and two for free ATP, one with low and another with high affinity. Citrate would compete with free ATP for the same sites. The interaction of free ATP with the high affinity site activated the enzyme, whereas its interaction with the low affinity site would lead to an inhibition.     

Abundance in Sewage of Bacteriophages That Infect Escherichia coli O157:H7 and That Carry the Shiga Toxin 2 Gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Modification of cys-128 of pig kidney fructose 1,6-bisphosphatase with different thiol reagents: Size dependent effect on the substrate and fructose-2,6-bisphosphate interaction

Treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide was shown to abolish the inhibition by fructose 2,6-bisphosphate, which also protected the enzyme against this chemical modification [Reyes, A., Burgos, M. E., Hubert, E., and Slebe, J. C. (1987),J. Biol. Chem. 262, 8451–8454]. On the basis of these results, it was suggested that a single reactive sulfhydryl group was essential for the inhibition. We have isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reagents with bulky groups are used to modify the protein at the reactive sulfhydryl [e.g., N-ethylmaleimide or 5,5-dithiobis-(2-nitrobenzoate)], most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller groups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviolet difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerable reduction in the affinity of the enzyme for fructose 2,6-bisphosphate while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the enzyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although this residue does not seem to be essential for the inhibition to occur. The results also suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate may interact with the enzyme in a different way.     

Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export

Background

The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines.

Methodology/Principal Findings

To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies.

Conclusions/Significance

The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.     

Digit length may reveal unusual breeding behaviour in a seabird

The hormonal environment experienced during prenatal development may affect adult phenotype and behaviour. Digit lengths may provide an estimate of steroid levels encountered during embryonic development in humans and other vertebrates. Finger patterns in humans have been shown to reveal sexual orientation or cooperative behaviour. We explored individual breeding behaviour in a monogamous seabird, the Balearic shearwater Puffinus mauretanicus and unexpectedly detected some cooperative breeders. Furthermore, we show evidence of correlation between digit lengths and cooperative breeding in this species. Additionally, we suggest that the first digit could be a possible indicator of prenatal steroid levels. These results are the starting point for further tests of the hypothesis that first digit length is an indicator of prenatal hormone levels in other vertebrate species. Moreover, these results may offer practical use in wild populations to study the implications of the changes in prenatal environment for adult social behaviour.     

Ecological and biogeographical inferences on two sympatric and enigmatic Andean cat species using genetic identification of faecal samples

The carnivore community of the altiplano ecosystem of the high Andes, including the Andean mountain cat (Leopardus jacobita) and pampas cat (Leopardus colocolo), is one of the least studied in the world. We determined the origin of 186 carnivore samples (184 faeces and two skulls) collected above 3000 m above sea level in northern Chile, including 33 from the Andean mountain cat and 75 from the pampas cat using diagnostic molecular genetic sequence variation. We determined for the first time food habits, habitat and physiographic associations, and general patterns of molecular genetic variation of the Andean mountain cat and the pampas cat in Chile. Both species had narrow dietary niches dominated by small rodents and there was a wide overlap in diet composition (0.82), suggesting low levels of prey partitioning between species. The mountain viscacha (Lagidium viscacia) made up a large proportion of the biomass of the diet of both species, especially for the Andean mountain cat (93.9% vs. 74.8% for the pampas cat), underscoring the importance of further research and conservation focus on this vanishing prey species. Although the probability of finding Andean mountain cat scats increased with altitude and slope, there was substantial geographical overlap in distribution between species, revealing that the pampas cat distribution includes high-altitude grassland habitats. The Andean mountain cat had relatively low levels of mitochondrial DNA (mtDNA) genetic variation (two mtDNA haplotypes) compared with the pampas cat (17 mtDNA haplotypes), suggestive of a distinct evolutionary history and relatively smaller historic populations. These insights will facilitate and provide tools and hypotheses for much-needed research and conservation efforts on these species and this ecosystem.     

Bridging the gap between plant and mammalian polyamine catabolism: a novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis

In contrast to animals, where polyamine (PA) catabolism efficiently converts spermine (Spm) to putrescine (Put), plants have been considered to possess a PA catabolic pathway producing 1,3-diaminopropane, Delta(1)-pyrroline, the corresponding aldehyde, and hydrogen peroxide but unable to back-convert Spm to Put. Arabidopsis (Arabidopsis thaliana) genome contains at least five putative PA oxidase (PAO) members with yet-unknown localization and physiological role(s). AtPAO1 was recently identified as an enzyme similar to the mammalian Spm oxidase, which converts Spm to spermidine (Spd). In this work, we have performed in silico analysis of the five Arabidopsis genes and have identified PAO3 (AtPAO3) as a nontypical PAO, in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N(1)-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (agmatine, cadaverine, and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C terminus, and it targets green fluorescence protein to peroxisomes when fused at the N terminus but not at the C terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.     

Molecular identification and functional characterization of the vitamin C transporters expressed by Sertoli cells

Vitamin C is an essential micronutrient for the development of male germ cells. In the gonad, the germ cells are isolated from the systemic circulation by the blood-testis barrier, which consists of a basal layer of Sertoli cells that communicate through an extensive array of tight junction complexes. To study the behavior of Sertoli cells as a first approach to the molecular and functional characterization of the vitamin C transporters in this barrier, we used the 42GPA9 cell line immortalized from mouse Sertoli cells. To date, there is no available information on the mechanism of vitamin C transport across the blood-testis barrier. This work describe the molecular identity of the transporters involved in vitamin C transport in these cells, which we hope will improve our understanding of how germ cells obtain vitamin C, transported from the plasma into the adluminal compartment of the seminiferous tubules. RT-PCR analyses revealed that 42GPA9 cells express both vitamin C transport systems, a finding that was confirmed by immunocytochemical and immunoblotting analysis. The kinetic assays using radioactive vitamin C revealed that both ascorbic acid (AA) transporters, SVCT1 and SVCT2, are functionally active. Moreover, the kinetic characteristics of dehydroascorbic acid (DHA) and 3-methylglucose (OMG) transport by 42GPA9 Sertoli cells correspond to facilitative hexose transporters GLUT1, GLUT2 and GLUT3 expressed in these cells. This data is consistent with the concept that Sertoli cells have the ability to take up vitamin C. It is an important finding and contributes to our knowledge of the physiology of male germ cells.