Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization

Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization. Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E. coli promoter. The transgenic E. coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E. coli, suggesting the synthesis of an active protein. The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species. The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E. coli through an unknown mechanism. The study elucidates a hitherto unknown possible function of PQQ in bacteria.     

The interactions of ATP, ADP, and inorganic phosphate with the sheep cardiac ryanodine receptor.

The effects of ATP, ADP, and inorganic phosphate (Pi) on the gating of native sheep cardiac ryanodine receptor channels incorporated into planar phospholipid bilayers were investigated. We demonstrate that ATP and ADP can activate the channel by Ca2+-dependent and Ca2+-independent mechanisms. ATP and ADP appear to compete for the same site/s on the cardiac ryanodine receptor, and in the presence of cytosolic Ca2+ both agents tend to inactivate the channel at supramaximal concentrations. Our results reveal that ATP not only has a greater affinity for the adenine nucleotide site/s than ADP, but also has a greater efficacy. The EC50 value for channel activation is approximately 0.2 mM for ATP compared to 1.2 mM for ADP. Most interesting is the fact that, even in the presence of cytosolic Ca2+, ADP cannot activate the channel much above an open probability (Po) of 0.5, and therefore acts as a partial agonist at the adenine nucleotide binding site on the channel. We demonstrate that Pi also increases Po in a concentration and Ca2+-dependent manner, but unlike ATP and ADP, has no effect in the absence of activating cytosolic [Ca2+]. We demonstrate that Pi does not interact with the adenine nucleotide site/s but binds to a distinct domain on the channel to produce an increase in Po.     

Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton

The long bones of the vertebrate appendicular skeleton arise from initially continuous condensations of mesenchymal cells that subsequently segment and cavitate to form discrete elements separated by synovial joints. Little is known, however, about the molecular mechanisms of joint formation. We present evidence that Wnt-14 plays a central role in initiating synovial joint formation in the chick limb. Wnt-14 is expressed in joint-forming regions prior to the segmentation of the cartilage elements, and local misexpression of Wnt-14 induces morphological and molecular changes characteristic of the first steps of joint formation. Induction of an ectopic joint-like region by Wnt-14 suppresses the formation of the immediately adjacent endogenous joint, potentially providing insight into the spacing of joints.     

LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice

Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::β-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice.     

A mathematical model in immunoregulation where the spleen has a pivotal role.

A simple mathematical model is introduced to investigate collaborative interactions between thymus-derived T and bursal-influenced B lymphocytes in the presence of specific antigen, intravenously presented. Such encounters are assumed to lead to humoral antibody response, and antigen interacting to produce effector T cells in the absence of B cells is assumed to produce cell mediated immunity. The model is used further to consider (i) the resultant distribution of effector T cells, (ii) the effect of splenectomy on collaborative T-B cell interactions, and (iii) recent data relating to portal cirrhosis, sickle cell anemia, malignant lymphomas, and diseases involving an impaired thymic function.It is concluded that for soluble antigen, such T-B cell encounters normally predominate in the spleen, as compared with the lymph nodes. Developing principles of immunoregulation may be applied to the theoretical basis of the model.     

Regulatory role of phosphate and other anions in transport of ADP and ATP by Rickettsia prowazekii.

ADP and ATP were transported in Rickettsia prowazekii by an obligate exchange system without prior hydrolysis. The uptake of ATP and ADP by the obligate exchange system in R. prowazekii was dependent upon the anionic composition of the medium. The rate of transport of ATP was about three times greater than that of ADP in the absence of anions, and the rates of transport of both were about doubled by a variety of anions. However, phosphate anions were able to stimulate greatly the uptake of ADP so that in the presence of these anions, the uptake of ATP and that of ADP were about equal. Millimolar concentrations of anions were required to elicit the stimulation of ADP and ATP transport. The ADP-dependent efflux of ADP and ATP was also greatly stimulated by phosphate anions. The stimulation of ADP and ATP transport required that the anions be present in the external medium, as preincubation of the rickettsiae with phosphate anions was neither necessary nor sufficient. The competitive inhibition of ATP uptake by ADP required phosphate anions, indicating that phosphate anions increased the affinity of ADP for the transport system. The role of phosphate in the regulation of ATP and ADP exchange and its significance are discussed.     

The nature of absorbance changes following the addition of ADP to mitochondria: ATP synthesis from intramitochondrial inorganic phosphate

The transient absorbance increase induced by ADP in phosphate-loaded respiring mitochondria becomes stable and greatly amplified by inhibitors of phosphate transport. The absorbance changes are sensitive to oligomycin and to aurovertin and their extent is proportional to the amount of ADP added. Simultaneously with the ADP-dependent increase in absorbance the inorganic phosphate and K⁺-ion content of the matrix decreases. It is concluded that the optical change reflects contraction of the matrix compartment secondary to intramitochondrial solute changes.     

S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase.

All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( approximately 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l-(35)S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( approximately 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.     

Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events.     

Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury

Acute lung injury (ALI) is a severe illness with excess mortality and no specific therapy. In its early exudative phase, neutrophil activation and accumulation in the lung lead to hypoxemia, widespread tissue damage, and respiratory failure. In clinical trials, inhibition of proinflammatory mediators has not proven effective. In this study, we pursued a new investigative strategy that emphasizes mediators promoting resolution from lung injury. A new spontaneously resolving experimental murine model of ALI from acid aspiration was developed to identify endogenous proresolving mechanisms. ALI increased cyclooxygenase 2 (COX-2) expression in murine lung. Selective pharmacologic inhibition or gene disruption of COX-2 blocked resolution of ALI. COX-2-derived products increased levels of the proresolving lipid mediators lipoxin A4 (LXA4) and, in the presence of aspirin, 15-epi-LXA4. Both LXA4 and 15-epi-LXA4 interact with the LXA4 receptor (ALX) to mediate anti-inflammatory actions. ALX expression was markedly induced by acid injury and transgenic mice with increased ALX expression displayed dramatic protection from ALI. Together, these findings indicate a protective role in ALI for COX-2-derived mediators, in part via enhanced lipoxin signaling, and carry potential therapeutic implications for this devastating clinical disorder.     

Effects of the ATP, ADP and inorganic phosphate on the transport rate of the Na+,K+-pump

(Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. In the reconstituted system the pump can be activated by adding ATP to the external medium. ATP-driven potassium extrusion by the Na+,K+-pump was studied using a voltage-sensitive dye in the presence of valinomycin. ADP strongly reduced the turnover rate of the pump with a concentration for half-maximal inhibition of cD,1/2 = 0.1 mM. cD,1/2 was found to be virtually independent of ATP concentration, indicating that the inhibition is non-competitive with respect to ATP. The non-competitive inhibition by ADP can be explained on the basis of the Post-Albers reaction cycle of the Na+,K+-pump, assuming that the main action of ADP is the reversal of the phosphorylation step. A similar 'product inhibition' was observed with inorganic phosphate, but at much higher concentrations (cP,1/2 = 14 mM).     

Pax8 plays a pivotal role in regulation of cardiomyocyte growth and senescence

Congenital heart disease (CHD) is a worldwide health problem, particularly in young populations. In spite of the advancement and progress in medical research and technology, the underlying causative factors and mechanisms of CHD still remain unclear. Bone morphogenetic protein receptor IA (ALK3) mediates the development of ventricular septal defect (VSD). We have recently found that paired box gene 8 (Pax8) may be the downstream molecule of ALK3. Paired box gene 8 plays an essential role in VSD, and apoptosis and proliferation imbalance leads to septal dysplasia. Recent studies have also disclosed that cellular senescence also participates in embryonic development. Whether programmed senescence exists in cardiac organogenesis has not ever been reported. We hypothesized that together with various biological processes, such as apoptosis, enhanced cellular senescence may occur actively in the development of Pax8 null mice murine hearts. In H9C2 myogenic cells, Pax8 overexpression can rescue caspase‐dependent apoptosis induced by ALK3 silencing. Senescent cells and senescence‐associated mediators in Pax8 knockout hearts increased compared with the wild‐type ones in an age‐dependent manner. These results suggest that Pax8 maybe the downstream molecule of ALK3, it mediates the murine heart development perhaps via cellular senescence, which may serve as a mechanism that compensates for the cell loss via apoptosis in heart development.     

Interdependence between mitochondrial matrix volume and the extramitochondrial ratio of [ATP]/[ADP] [inorganic phosphate].

ATP or combinations of ATP with EDTA and EGTA can act as chelators to support succinate-driven, phosphate-requiring expansion of mitochondrial inner membrane-matrices. Contraction of these swollen mitochondria can be induced with antimycin, MgCl₂ and ADP. The magnitude of ADP-induced contraction of mitochondria, swollen in the presence of ATP, is dependent on [ADP] and may be altered by the extramitochondrial concentrations of both P_i and ATP. In fact, the extent of contraction (+ΔA₅₂₀) is a linear function of the thermodynamic parameter, ?ΔGp (free energy of hydrolysis of ATP), provided excessive concentrations of reactants are not present and the extents of matrix swelling are similar ($\text{e.g.}\text{ΔA}{}_{520}$ is about 0.250) before starting contraction with ADP.     

Measurement temperature plays a pivotal role in the distribution of erythrocyte deformability after LPS

Reductions in red blood cell membrane deformability (RBC(D)) may perturb microcirculatory blood flow and impair tissue O(2)-availability. We investigated the effect of assay temperature on the distribution of RBC(D) in endotoxin (LPS) incubated and control RBCs. Fresh blood from healthy rats was incubated with and without the presence of LPS for 6 hrs. An index of red blood cell membrane deformability, delta, was measured via the micropipette aspiration technique at 25 degrees C and 37 degrees C at 0, 2 and 6 hrs of incubation. The ATP content of RBC was measured by the luciferin-luciferase technique. At 25 degrees C, LPS caused a significant decrease in mean delta after 2 and 6 hours incubation compared to controls (-10.0%, p=0.03 and -24.0%, p=0.03, respectively) characterized by a left shift in the distribution (skewness: -1.4). However, at 37 degrees C a significant decrease in delta was only detected after 6 hrs of LPS incubation (-13.8%, p=0.01, compared to -5.1%, p=0.7 at 2 hours) and lacked the left shifted distribution (skewness: 0.2). No significant difference in ATP content of RBCs was observed between groups. We have shown that LPS incubation results in a significant decrease in RBC(D) and that room temperature measurement of physical membrane properties may exaggerate the differences between normal and perturbed RBCs.     

Synthesis of ATP and free radicals in an aqueous solution, containing NADH, riboflavin, ADP and inorganic phosphate

pH-dependence of initial (admixture) adenosine triphosphate (ATP) changes (ATP synthesis and hydrolysis) was studied for aerated and deaerated aqueous solutions during the incubation of adenosine diphosphate (ADP) and inorganic phosphate (Pi) in the presence of reduced nicotinamide adenine dinucleotide (NADH) and riboflavin (Rf). The preferential pH regions were indicated both for ATP synthesis and ATP hydrolysis (pH less than pH 5.4, 5.9 divided by pH 6.5 and pH greater than 6.6, pH 5.5 divided by pH 5.8 respectively). 'Free radical content measurements were paralleled by ESR technique. On the basis of the obtained results it was assumed that a part of ESP signal attributed to ADP radicals was increased during ATP synthesis.     

EGFR plays a pivotal role in the regulation of polyamine-dependent apoptosis in intestinal epithelial cells

Intracellular polyamine synthesis is regulated by the enzyme ornithine decarboxylase (ODC), and its inhibition by -difluromethylornithine (DFMO), confers resistance to apoptosis. We have previously shown that DFMO leads to the inhibition of de novo polyamine synthesis, which in turn rapidly activates Src, STAT3 and NF-κB via integrin β3 in intestinal epithelial cells. One mechanism to explain these effects involves the activation of upstream growth factor receptors, such as the epidermal growth factor receptor (EGFR). We therefore hypothesized that EGFR phosphorylation regulates the early response to polyamine depletion. DFMO increased EGFR phosphorylation on tyrosine residues 1173 (pY1173) and 845 (pY845) within 5 min. Phosphorylation declined after 10 min and was prevented by the addition of exogenous putrescine to DFMO containing medium. Phosphorylation of EGFR was concomitant with the activation of ERK1/2. Pretreatment with either DFMO or EGF for 1 h protected cells from TNF-/CHX-induced apoptosis. Exogenous addition of polyamines prevented the protective effect of DFMO. In addition, inhibition of integrin β3 activity (with RGDS), Src activity (with PP2), or EGFR kinase activity (with AG1478), increased basal apoptosis and prevented protection conferred by either DFMO or EGF. Polyamine depletion failed to protect B82L fibroblasts lacking the EGFR (PRN) and PRN cells expressing either a kinase dead EGFR (K721A) or an EGFR (Y845F) mutant lacking the Src phosphorylation site. Conversely, expression of WT-EGFR (WT) restored the protective effect of polyamine depletion. Fibronectin activated the EGFR, Src, ERKs and protected cells from apoptosis. Taken together, our data indicate an essential role of EGFR kinase activity in MEK/ERK-mediated protection, which synergizes with integrin β3 leading to Src-mediated protective responses in polyamine depleted cells.     

Mitochondrial ATP synthase. Crystal structure of the catalytic F1 unit in a vanadate-induced transition-like state and implications for mechanism

ATP synthesis from ADP, P(i), and Mg2+ takes place in mitochondria on the catalytic F1 unit (alpha3beta3gammedeltaepsilon) of the ATP synthase complex (F0F1), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F1, the P-loop (amino acid residues 156GGAGVGKT163) contributes to substrate binding at the subunit catalytic sites. Here, we report the first transition state-like structure of F1 (ADP.V(i).Mg.F1) from rat liver that was crystallized with the phosphate (P(i)) analog vanadate (VO(3-)4 or V(i)). Compared with earlier "ground state" structures, this new F1 structure reveals that the active site region has undergone significant remodeling. P-loop residue alanine 158 is located much closer to V(i) than it is to P(i) in a previous structural model. No significant movements of P-loop residues of the subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, thus facilitating the dehydration of ADP and P(i). This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of V(i) (Ko, Y. H., Bianchet, M., Amzel, L. M., and Pedersen, P. L. (1997) J. Biol. Chem. 272, 18875-18881; Ko, Y. H., Hong, S., and Pedersen, P. L. (1999) J. Biol. Chem. 274, 28853-28856).     

Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania. Characterization of gene deletion mutants

The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and Deltaarg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The Deltaarg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the Deltaarg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the Deltaarg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.