Escherichia coli F1-ATPase can use GTP-nonchaseable bound adenine nucleotide to synthesize ATP in dimethyl sulfoxide.

Escherichia coli F1-ATPase contained 3 mol of tightly-bound adenine nucleotide/mol enzyme. A further 3 mol could be loaded by incubation of the enzyme with ATP. The unloaded enzyme was designated as a F1[2,1] type on the basis of the ability of GTP to displace 1 mol of adenine nucleotide/mol of F1 [Kironde, F.A.S., & Cross, R.L. (1986) J. Biol. Chem. 261, 12544-12549]. The loaded enzyme was designated F1[3,3] since GTP could displace 3 of the 6 mol of bound adenine nucleotide/mol of F1. Incubation of F1[2,1], F1[2,0], and F1[3,0] with phosphate in the presence of 30% (v/v) dimethyl sulfoxide led to the synthesis of ATP from endogenous bound ADP. Hydrolysis of newly synthesized ATP occurred on transfer of the F1 from 30% (v/v) dimethyl sulfoxide to an entirely aqueous medium. Thus, synthesis and hydrolysis of ATP can occur at GTP-nonchaseable adenine nucleotide binding sites, and these sites in dimethyl sulfoxide are not necessarily equivalent to noncatalytic sites.     

A Comparison Between Bonobos and Chimpanzees: A Review and Update

Chimpanzees (Pan troglodytes) and bonobos (P. paniscus) are our closest living relatives, with the human lineage diverging from the Pan lineage only around five to seven Mya, but possibly as early as eight Mya.^1–2 Chimpanzees and bonobos even share genetic similarities with humans that they do not share with each other.² Given their close genetic relationship to humans, both Pan species represent crucial living models for reconstructing our last common ancestor (LCA) and identifying uniquely human features. Comparing the similarities and differences of the two Pan is thus essential for constructing balanced models of human evolution.³     

Biochemical changes in prostanoids and cerebral expression of cyclooxygenase (COX)-1 and COX-2 during morphine sulfate infusion in the newborn piglet

To examine the biochemical regulation of morphine sulfate (MS) on prostanoid synthesis, conscious newborn piglets received a bolus dose of 100 microg/kg followed by a continuous infusion dose of 100 microg/kg/h. The control group received equivalent volume bolus and continuous infusion of 5% dextrose. Blood samples were drawn from the femoral artery and sagittal sinus vein before, during and after infusion for measurement of prostanoids. The expression of mRNAs encoding cyclooxygenases (COX)-1 and -2 in the brainstem, thalamus, cortex, and cerebellum of the newborn piglets were also examined. Systemic PGE2 levels declined substantially during and post MS infusion (p < 0.01), whereas sagittal sinus vein PGE2 levels increased following the bolus dose (p < 0.01) and at 4 h of continuous infusion (p < 0.01). MS infusion did not affect systemic 6-ketoPGF1alpha levels, however, in the cerebral circulation 6-ketoPGF1alpha levels increased 146% (p < 0.01) following the bolus dose and remained elevated throughout the infusion and post infusion times. Systemic TxB2 levels increased transiently at 4 h (p < 0.01) and sagittal sinus vein TxB2 increased at 0.5 and 1 h (p < 0.01) during continuous infusion. RT-PCR assays revealed a 1.5- (p < 0.001) to 4-fold (p < 0.001) increased expression of COX-1 mRNA in the MS-infused brain samples. In contrast, no differences in COX-2 mRNA were detected between the groups. These data imply that MS may have significant effects on prostanoid synthesis in the newborn. The data further show that the MS-induced prostanoid responses appear to be mediated via COX-1.     

Leber's hereditary optic neuropathy (LHON) pathogenic mutations induce mitochondrial-dependent apoptotic death in transmitochondrial cells incubated with galactose medium

Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochrome c into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria.     

Histidine ligand protonation and redox potential in the rieske dioxygenases: role of a conserved aspartate in anthranilate 1,2-dioxygenase

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be approximately 100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.     

Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

In vitro response of prostaglandin E2 receptor (EP3) in the term pregnant rat uterus and cervix to misoprostol

We examined and compared the in vitro effects of misoprostol (synthetic prostaglandin E1 (PGE1) analogue) on prostaglandin E2 (PGE2) secretion and EP3 receptor mRNA expression in the pregnant rat myometrium and cervix at 19 days gestation. Myometrial and cervical tissue samples were exposed to media with or without misoprostol (50 or 100 pg/ml) and incubated for 15 and 30 min, and 1, 3, 6, 12, and 24 h. Media and tissue samples were collected for quantification of PGE2 and mRNA expression of rEP3alpha and rEP3beta receptor, respectively. PGE2 secretion increased (P < or = 0.05) in the myometrium exposed to 50 and 100 pg/ml misoprostol. Cervical PGE2 secretion increased following exposure to the 100 pg/ml dose only. In the myometrium, 50 and 100 pg/ml misoprostol induced elevations in rEP3alpha and rEP3beta receptor mRNA expression. rEP3alpha and rEP3beta receptor mRNA expression in the cervix was not different from controls. These data demonstrate that the EP3 receptor is differentially expressed in the myometrium and cervix in response to misoprostol. This may account for the ability of misoprostol to stimulate the myometrium when administered for cervical ripening.     

Bioenergetics shapes cellular death pathways in Leber's hereditary optic neuropathy: a model of mitochondrial neurodegeneration

Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically. In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress. The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways.