Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO₂ reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.Phosphorus (P) is an important nutrient for all living organisms. The predominant forms of phosphorus in biological systems are inorganic phosphate and its organic esters and acid anhydrides in which P is at its highest oxidation state (+V). The P requirements of living cells can be fulfilled with phosphate in various forms, including reduced organic and inorganic phosphorus compounds (23). Several aerobic bacteria were shown to be able to oxidize hypophosphite (+I) and phosphite (+III) to phosphate (+V) and to incorporate the last into their biomass (5, 15-17, 31, 34). Phosphite can also be oxidized under anaerobic conditions, as shown for an anaerobic Bacillus strain (7) and for Pseudomonas stutzeri which can use phosphite under denitrifying conditions (17, 21). The only bacterium known to oxidize phosphite as the sole source of electrons in lithoautotrophic energy metabolism is Desulfotignum phosphitoxidans (24, 25).Three different metabolic pathways for the use of phosphite as a single P source have been characterized so far. Two of them were discovered and characterized with Escherichia coli and one with Pseudomonas stutzeri. The first pathway in E. coli is mediated by the enzyme carbon phosphorus lyase (C-P lyase), and the second one by the alkaline phosphatase encoded by phoA (16, 34). This alkaline phosphatase not only hydrolyzes phosphate esters but also hydrolyzes phosphite to phosphate and molecular hydrogen (32). This is a particular property only of the E. coli alkaline phosphatase and is not observed with alkaline phosphatases of other bacteria. The third pathway is encoded by the ptxABCDE gene cluster in P. stutzeri (17). In this system, phosphite is transported into the cell by a binding protein-dependent phosphite transporter at the expense of ATP (PtxABC). Phosphite is oxidized by a phosphite:NAD⁺ oxidoreductase (encoded by ptxD), a new member of the 2-hydroxy acid dehydrogenases (8). The ptx operon of P. stutzeri is regulated in response to phosphate starvation by the two-component regulatory system phoBR (28, 29). Furthermore, in Alcaligenes faecalis WM2072, another gene cluster involved in hypophosphite and phosphite uptake and oxidation was characterized: the htxABCD-ptxDE locus (31). The htxABCD-ptxDE genes and their products in A. faecalis WM 2072 have high nucleotide and amino acid sequence identities with those found in the htx and ptx operons in P. stutzeri WM88, which are required for the oxidation of hypophosphite and phosphite, respectively. This unique genetic arrangement of hypophosphite- and phosphite-oxidizing genes in A. faecalis WM2072 suggests a horizontal gene transfer and an ancient evolution of phosphite oxidation.The diversity of pathways used for assimilatory phosphite oxidation and the fact that D. phosphitoxidans is so far the only bacterium known to use phosphite as an electron source caused us to investigate the phosphite uptake and oxidation gene cluster of this bacterium. The aims of our study were (i) to establish enzymatic assays for measurement of phosphite oxidation activity in cell extracts, (ii) to identify the genes involved in phosphite uptake and oxidation, and (iii) to characterize these genes physiologically.     

The roles of IL-12 and IL-23 in CD8 T cell-mediated immunity against Listeria monocytogenes: Insights from a DC vaccination model

Listeria monocytogenes infection induces a strong inflammatory response characterized by the production of IL-12 and IFN-γ and protective immunity against this pathogen is dependent on CD8⁺ T cells (CTL). Recent studies have suggested that these inflammatory cytokines affect the rate of memory CD8⁺ T cell generation as well as the number of short-lived effector cells generated. The role of the closely related cytokine, IL-23, in this response has not been examined. We hypothesized that IL-12 and IL-23 produced by dendritic cells collectively enhance the generation and function of memory cells. To test this hypothesis, we employed a DC vaccination approach. Mice lacking IL-12 and IL-23 were vaccinated with wild-type (WT), IL-12^−/−, or IL-12/23^−/− DC and protection to Lm was monitored. Mice vaccinated with WT and IL-12^−/− DC were resistant to lethal challenge with Lm. Surprisingly, mice vaccinated with IL-12/23^−/− DC exhibited significantly reduced protection when challenged. Protection correlated with the relative size of the memory pools generated. In summary, these data indicate that IL-23 can partially compensate for the lack of IL-12 in the generation protective immunity against Lm.     

A survey of formal methods for determining the centre of rotation of ball joints

The determination of an accurate centre of rotation (CoR) from segment marker positions is of interest across a wide range of applications, but particularly for clinical gait analysis and for estimating the hip joint centre during surgical intervention of the knee, for limb alignment purposes. For the first time in this survey of formal methods, we classify, analyse and compare different methods (geometric, algebraic, bias compensated algebraic, and Pratt sphere fit methods, as well as the centre transformation technique, the Holzreiter approach, the helical pivot technique, the Schwartz transformation techniques, the minimal amplitude point method and the Stoddart approach) for the determination of spherical joint centres from marker position data. In addition, we propose a new method, the symmetrical CoR estimation or SCoRE, in which the coordinates of the joint centre must only remain constant relative to each segment, thus not requiring the assumption that one segment should remain at rest. For each method, 1000 CoR estimations were analysed with the application of isotropic, independent and identically distributed Gaussian noise (standard deviation 0.1cm) to each of the marker positions, to all markers on the segment simultaneously and the two in combination. For the test conditions used here, most techniques were capable of determining the CoR to within 0.3 cm, as long as the spherical range of motion (RoM) of the joint was 45 degrees or more. Under the most stringent conditions tested, however, the SCoRE was capable of best determining the CoR, to within approximately 1.2mm with a RoM of 20 degrees . The correct selection and application of these methodologies should help improve the accuracy of surgical navigation and clinical kinematic measurement.     

Detrimental effect of eicosapentaenoic acid supplementation on bone following ovariectomy in rats

Although several studies have reported a positive effect of n-3 essential fatty acids (EFAs) on bone density post-ovariectomy, the role of specific EFAs has yet to be fully elucidated. In this study, ovariectomised (OVX) rats were supplemented with 0.1 g (LOW) or 1.0 g (HIGH) of eicosapentaenoic acid (EPA)/kg body weight for 9 weeks. Bone mineral density (BMD), 25-hydroxyvitamin D(3) and plasma fatty acid profile were compared to those of OVX and sham animals fed a non-supplemented diet. BMD decreased significantly in all OVX (P<0.001) but not sham rats. There was no difference in BMD between the LOW group and OVX controls. BMD was significantly lower in the HIGH group compared to OVX and sham controls. 25-hydroxyvitamin D(3) levels were significantly higher in both the LOW and HIGH groups compared to OVX controls (P=0.0006 and 0.02, respectively). In conclusion, high-dose EPA supplementation exacerbated the effects of ovariectomy on BMD.     

Rap1A-deficient T and B cells show impaired integrin-mediated cell adhesion

Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1-/- cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.     

Novel frequenin-modulated Ca2+-signaling membrane guanylate cyclase (ROS-GC) transduction pathway in bovine hippocampus

Frequenin is a member of the neuronal Ca²⁺ sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca²⁺-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca²⁺ myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca²⁺ myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca²⁺-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca²⁺-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.     

Inflammatory phase of bone healing initiates the regenerative healing cascade

Bone healing commences with an inflammatory reaction which initiates the regenerative healing process leading in the end to reconstitution of bone. An unbalanced immune reaction during this early bone healing phase is hypothesized to disturb the healing cascade in a way that delays bone healing and jeopardizes the successful healing outcome. The immune cell composition and expression pattern of angiogenic factors were investigated in a sheep bone osteotomy model and compared to a mechanically-induced impaired/delayed bone healing group. In the impaired/delayed healing group, significantly higher T cell percentages were present in the bone hematoma and the bone marrow adjacent to the osteotomy gap when compared to the normal healing group. This was mirrored in the higher cytotoxic T cell percentage detected under delayed bone healing conditions indicating longer pro-inflammatory processes. The highly activated periosteum adjourning the osteotomy gap showed lower expression of hematopoietic stem cell markers and angiogenic factors such as heme oxygenase and vascular endothelial growth factor. This indicates a deferred revascularization of the injured area due to ongoing pro-inflammatory processes in the delayed healing group. Results from this study suggest that there are unfavorable immune cells and factors participating in the initial healing phase. In conclusion, identifying beneficial aspects may lead to promising therapeutical approaches that might benefit further by eliminating the unfavorable factors.     

Assessing population-level variation in the mitochondrial genome of <Emphasis Type="Italic">Euphausia superba</Emphasis> using 454 next-generation sequencing

The Antarctic krill (Euphausia superba Dana 1852) is widely distributed throughout the Southern Ocean, where it provides a key link between primary producers and upper trophic levels and supports a major commercial fishery. Despite its ecological and commercial importance, genetic population structure of the Antarctic krill remains poorly described. In an attempt to illuminate genetic markers for future population and phylogenetic analysis, five E. superba mitogenomes, from samples collected west of the Antarctic Peninsula, were sequenced using new 454 next-generation sequencing techniques. The sequences, of lengths between 13,310 and 13,326 base pairs, were then analyzed in the context of two previously-published near-complete sequences. Sequences revealed relatively well-conserved partial mitochondrial genomes which included complete sequences for 11 of 13 protein-coding genes, 16 of 23 tRNAs, and the large ribosomal subunit. Partial sequences were also recovered for cox1 and the small ribosomal subunit. Sequence analysis suggested that the cox2, nad5, and nad6 genes would be the best candidates for future population genetics analyses, due to their high number of variable sites. Future work to reveal the noncoding control region remains.     

Melatonin: neuritogenesis and neuroprotective effects in crustacean x-organ cells

Melatonin has both neuritogenic and neuroprotective effects in mammalian cell lines such as neuroblastoma cells. The mechanisms of action include receptor-coupled processes, direct binding and modulation of calmodulin and protein kinase C, and direct scavenging of free radicals. While melatonin is produced in invertebrates and has influences on their physiology and behavior, little is known about its mechanisms of action. We studied the influence of melatonin on neuritogenesis in well-differentiated, extensively-arborized crustacean x-organ neurosecretory neurons. Melatonin significantly increased neurite area in the first 24 h of culture. The more physiological concentrations, 1 nM and 1 pM, increased area at 48 h also, whereas the pharmacological 1 μM concentration appeared to have desensitizing effects by this time. Luzindole, a vertebrate melatonin receptor antagonist, had surprising and significant agonist-like effects in these invertebrate cells. Melatonin receptors have not yet been studied in invertebrates. However, the presence of membrane-bound receptors in this population of crustacean neurons is indicated by this study. Melatonin also has significant neuroprotective effects, reversing the inhibition of neuritogenesis by 200 and 500 μM hydrogen peroxide. Because this is at least in part a direct action not requiring a receptor, melatonin's protection from oxidative stress is not surprisingly phylogenetically-conserved.