Bxz1, a new generalized transducing phage for mycobacteria

We have isolated and characterized a new generalized transducing phage, Bxz1, from soil sampling at a neighboring Wildlife Preservation Park. The hosts of the phage, measured by the formation of plaques, include fast growing Mycobacterium smegmatis and Mycobacterium vaccae. Bxz1 is capable of transducing chromosomal markers, point mutations, and plasmids at frequencies ranging from 10(-8) to 10(-6) per plaque forming unit between strains of M. smegmatis. We also demonstrated cotransduction of a transposon insertion linked to a point mutation of the ndh gene.     

Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.     

Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide

In addition to the (Na⁺+K⁺)ATPase another P-ATPase, the ouabain-insensitive Na⁺-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg²⁺, MgATP and furosemide on the Na⁺-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na⁺-ATPase at pH 7.0 are: (a) K_0.5 for Na⁺=8.9±2.2 mM; (b) K_0.5 for MgATP=1.8±0.4 mM; (c) two sites for free Mg²⁺: one stimulatory (K_0.5=0.20±0.06 mM) and other inhibitory (I_0.5=1.1±0.4 mM); and (d) I_0.5 for furosemide=1.1±0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na⁺ (K_0.5=19.5±0.4) and MgATP (K_0.5=3.4±0.3 mM) but increases the apparent affinity for furosemide (0.18±0.02 mM) and Mg²⁺ (0.05±0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na⁺ (K_0.5=18.7±1.5 mM) and furosemide (I_0.5=3.04±0.57 mM) but does not change the apparent affinity to MgATP and Mg²⁺. The data presented in this paper indicate that the modulation of the Na⁺-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg²⁺ and MgATP complex may play an important role in the Na⁺-ATPase physiological regulatory mechanisms.     

Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis

BACKGROUND: 3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg(2+) for activity. RESULTS: The first three-dimensional structure of the enzyme was determined at 1.4 A resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an alpha + beta fold having a complex linkage of beta strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks. CONCLUSIONS: A proposed active site was identified on the basis of amino acid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, which may be used for recognizing the phosphate group of the substrate, the active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg(2+) cofactor within the active site.     

The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria

Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate- and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate- or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H₂O₂ release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells.     

Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis

The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson’s disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA.     

Deletion of late cornified envelope genes,LCE3C_LCE3B-del,is not associated with psoriatic arthritis in Tunisian patients

A deletion of two genes from the late cornified envelope (LCE), LCE3B and LCE3C within epidermal differentiation complex on chromosome 1 was shown to be associated with both psoriasis and psoriatic arthritis (PsA) in several populations. To assess whether this deletion may contribute to the genetic predisposition to PsA in Tunisia, a total of 73 patients with PsA and 120 healthy matched controls were screened for the deletion, LCE3C_LCE3B-del, and its tag SNP, rs4112788. We also evaluated a possible relationship between PSORS1 and LCE3C_LCE3B-del through genotyping two proxy markers to HLA-C (rs12191877 and rs2073048). Our results did not provide evidence for association between the LCE3C_LCE3B-del nor the rs4112788 and the PsA. Similarly, no significant epistatic effect was observed. Our data suggest that The LCE deletion, previously identified in patients with psoriasis, is not of a major importance in the development of PsA in Tunisian patients supporting the current perception that different genetic risk factors contribute to skin and joint disease. However, these results need to be confirmed by additional large-scale studies of Tunisian PsA patients and controls.     

A 7-day oral supplementation with branched-chain amino acids was ineffective to prevent muscle damage during a marathon

The aim of this study was to determine the effectiveness of a 7-day oral supplementation with branched-chain amino acids (BCAA) to prevent muscle damage during a marathon. Forty-six experienced runners were randomly divided into two groups, one with BCAA supplementation (n = 25, supplemented with 5 g day^?1 of powdered 1:0.5:0.5 leucine:isoleucine:valine, during the 7 days prior to the competition) and the other as a control group (n = 21, supplemented with an isocaloric placebo). Before the marathon race and within 3 min of finishing, leg muscle power was measured with a maximal countermovement jump and a urine sample was obtained. During the race, running pace was measured by means of a time-chip. Myoglobin concentration was determined in the urine samples as an indirect marker of muscle damage. A visual analog scale (0–10 points) was used to assess leg muscle pain during the race. In the BCAA group, the mean running pace during the marathon was similar to the control group (3.3 ± 0.4 vs. 3.3 ± 0.5 m s^?1, respectively, 0.98). The pre- to post-race reduction in muscle power was similar in both BCAA and control groups (?23.0 ± 16.1 vs. ?17.3 ± 13.8 %, P = 0.13). Post-race urine myoglobin concentration was similar in both BCAA and control groups (5.4 ± 7.5 vs. 4.5 ± 8.6 μg mL^?1, P = 0.70). Finally, there were no differences between groups in the perceived muscle pain during the race (6 ± 1 vs. 5 ± 1 points, P = 0.80). A 7-day supplementation of BCAA (5 g day^?1) did not increase the running performance during a marathon. Furthermore, BCAA supplementation was ineffective to prevent muscle power loss, muscle damage or perceived muscle pain during a marathon race.     

Ecological and evolutionary patterns of freshwater maturation in Pacific and Atlantic salmonines

Reproductive tactics and migratory strategies in Pacific and Atlantic salmonines are inextricably linked through the effects of migration (or lack thereof) on age and size at maturity. In this review, we focus on the ecological and evolutionary patterns of freshwater maturation in salmonines, a key process resulting in the diversification of their life histories. We demonstrate that the energetics of maturation and reproduction provides a unifying theme for understanding both the proximate and ultimate causes of variation in reproductive schedules among species, populations, and the sexes. We use probabilistic maturation reaction norms to illustrate how variation in individual condition, in terms of body size, growth rate, and lipid storage, influences the timing of maturation. This useful framework integrates both genetic and environmental contributions to conditional strategies for maturation and, in doing so, demonstrates how flexible life histories can be both heritable and subject to strong environmental influences. We review evidence that the propensity for freshwater maturation in partially anadromous species is predictable across environmental gradients at geographic and local spatial scales. We note that growth is commonly associated with the propensity for freshwater maturation, but that life-history responses to changes in growth caused by temperature may be strikingly different than changes caused by differences in food availability. We conclude by exploring how contemporary management actions can constrain or promote the diversity of maturation phenotypes in Pacific and Atlantic salmonines and caution against underestimating the role of freshwater maturing forms in maintaining the resiliency of these iconic species.