Mitochondrial and cell-surface F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase in innate and acquired cardioprotection

Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent advances in F₀F₁ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F₀F₁ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F₀F₁ATPsynthase regulation by the inhibitory protein IF₁ in heart preconditioning strategies; ii) the structure and function of mitochondrial F₀F₁ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult; iii) the external location and characterization of plasma membrane F₀F₁ ATP synthase in search for possible actors of its regulation, such as IF₁ and calmodulin, at cell surface.     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.     

Changes in S1P1 and S1P2 expression during embryonal development and primitive endoderm differentiation of F9 cells

Sphingosine 1-phosphate (S1P) is a ligand for S1P family receptors (S1P(1)-S1P(5)). Of these receptors, S1P(1), S1P(2), and S1P(3) are ubiquitously expressed in adult mice, while S1P(4) and S1P(5) are tissue specific. However, little is known of their expression during embryonal development. We performed Northern blot analyses in mouse embryonal tissue and found that such expression is developmentally regulated. We also examined the expression of these receptors during primitive endoderm (PrE) differentiation of mouse F9 embryonal carcinoma (EC) cells, a well-known in vitro endoderm differentiation system. S1P(2) mRNA was abundantly expressed in F9 EC cells, but little S1P(1) and no S1P(3), S1P(4), or S1P(5) mRNA was detectable. However, S1P(1) mRNA expression was induced during EC-to-PrE differentiation. Studies using small interference RNA of S1P(1) indicated that increased S1P(1) expression is required for PrE differentiation. Thus, S1P(1) may play an important function in PrE differentiation that is not substituted for by S1P(2).     

Manipulating the Length of the <Emphasis Type="Italic">b</Emphasis> Subunit F<Subscript>1</Subscript> Binding Domain in F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP Synthase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The peripheral stalk of F₁F₀ ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F₀ to the top of the F₁ sector. The stalk serves as the stator necessary for holding F₁ against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F₁. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F₁F₀ complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F₁F₀ ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F₁F₀ complex by a normal b subunit.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> purinoceptors in the rat enteric nervous system

Adenosine 5-triphosphate receptors are known to be involved in fast excitatory postsynaptic currents in myenteric neurons of the digestive tract. In the present study, the distribution of P2X₂ and P2X₃ receptor mRNA was examined by in situ hybridisation while P2X₂ and P2X₃ receptor protein was localised by immunohistochemical methods. In addition, P2X₂ and P2X₃ receptors were colocalised with calbindin and calretinin in the myenteric and submucosal plexus. P2X₂- and P2X₃-immunoreactive neurons were found in the myenteric and submucosal plexuses throughout the entire length of the rat digestive tract from the stomach to the colon. Approximately 60%, 70% and 50% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 56% and 45% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₂ receptor. Approximately 10%, 2% and 15% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 62% and 40% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₃ receptor. Double-labelling studies showed that about 10–25% of the neurons with P2X₂ immunoreactivity in myenteric plexus and 30–50% in the submucosal plexus were found to express calbindin or calretinin. About 80% of the neurons with P2X₃ receptor immunoreactivity in the myenteric plexus and about 40% in the submucosal plexus expressed calretinin. Approximately 30–75% of the neurons with P2X₃ receptor immunoreactivity in the submucosal plexus expressed calbindin, while none of them were found to express calbindin in the myenteric plexus.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Xylem parenchyma cells deliver the H<Subscript>2</Subscript>O<Subscript>2</Subscript> necessary for lignification in differentiating xylem vessels

Lignification in Zinnia elegans L. stems is characterized by a burst in the production of H₂O₂, the apparent fate of which is to be used by xylem peroxidases for the polymerization of p-hydroxycinnamyl alcohols into lignins. A search for the sites of H₂O₂ production in the differentiating xylem of Z. elegans stems by the simultaneous use of optical (bright field, polarized light and epi-polarization) and electron-microscope tools revealed that H₂O₂ is produced on the outer-face of the plasma membrane of both differentiating (living) thin-walled xylem cells and particular (non-lignifying) xylem parenchyma cells. From the production sites it diffuses to the differentiating (secondary cell wall-forming) and differentiated lignifying xylem vessels. H₂O₂ diffusion occurs mainly through the continuous cell wall space. Both the experimental data and the theoretical calculations suggest that H₂O₂ diffusion from the sites of production might not limit the rate of xylem cell wall lignification. It can be concluded that H₂O₂ is produced at the plasma membrane in differentiating (living) thin-walled xylem cells and xylem parenchyma cells associated to xylem vessels, and that it diffuses to adjacent secondary lignifying xylem vessels. The results strongly indicate that non-lignifying xylem parenchyma cells are the source of the H₂O₂ necessary for the polymerization of cinnamyl alcohols in the secondary cell wall of lignifying xylem vessels.     

Development of nerves expressing P2X<Subscript>3</Subscript> receptors in the myenteric plexus of rat stomach

Development of neurones and fibres expressing P2X₃ receptors in the myenteric plexus of rat stomach and coexistence of the P2X₃ receptor with calbindin, calretinin and NOS during postnatal development, were investigated with immunostaining methods. Extrinsic nerves expressing P2X₃ receptors appeared as early as E12 and were localised in the trunk and branches of the vagus nerve, which extended rapidly onto the whole rat stomach from E12 to E14. Intrinsic neurone cell bodies with P2X₃-immunoreactivity in the myenteric ganglia were first demonstrated postnatally at P1, and at P14, when the number of neurones expressing the P2X₃ receptor peaked at 45%. P2X₃ receptor-immunoreactivity decreased subsequently, and at P60 only about 11% were P2X₃-immunoreactive. Intraganglionic laminar nerve endings and intramuscular arrays were first demonstrated postnatally at P1 and P7, respectively. In the early postnatal days, there were many growth cone-like structures with strong P2X₃ immunostaining associated with these endings and arrays. Double-immunostaining showed that 9–15% of P2X₃-immunoreactive neurones in the gastric myenteric plexus expressed calbindin D-28 k only in the early postnatal days, while 14–21% of neurones from P1 to P60 increasingly expressed calretinin. About 20% of neurones with P2X₃ immunoreactivity coexpressed NOS throughout perinatal development.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Lysophosphatidic acid regulates inflammation-related genes in human endothelial cells through LPA1 and LPA3

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid (LPL), which regulates endothelial cells participating in inflammation processes via interactions with endothelial differentiation gene (Edg) family G protein-coupled receptors. In this study, we attempted to determine which LPA receptors mediate the inflammatory response in human endothelial cells. Introduction of siRNA against LPA₁ significantly suppressed LPA-induced ICAM-1 mRNA, total protein, and cell surface expressions, and subsequent U937 monocyte adhesion to LPA-treated human umbilical endothelial cells (HUVECs). By knock down of LPA₁ and LPA₃ in HUVECs, LPA-enhanced IL-1β mRNA expression was significantly attenuated. Moreover, LPA₁ and LPA₃ siRNA also inhibited LPA-enhanced IL-1-dependent long-term IL-8 and MCP-1 mRNA expression, and subsequent THP-1 cell chemotaxis toward LPA-treated HUVEC-conditioned media. These results suggest that the expression of LPA-induced inflammatory response genes is mediated by LPA₁ and LPA₃. Our findings suggest the possible utilization of LPA₁ or LPA₃ as drug targets to treat severe inflammation.     

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF<Subscript>1</Subscript>

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.