Inhibition of proinsulin biosynthesis and conversion by a putative insulin mediator

The phospho-oligosaccharide (POS), presumed to act at the postreceptor level as the insulin second messenger, was recently reported to inhibit glucose-stimulated insulin release from rat pancreatic islets. In the present study, POS was also found to inhibit glucose-stimulated proinsulin biosynthesis and conversion in rat islets. By comparison with prior findings on the effects of both exogenous insulin and anti-insulin serum upon proinsulin synthesis, these results argue against the view that insulin would normally exert a negative feedback control upon the biosynthetic and secretory activities of islet B-cells.     

Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B₁), and pyridoxine (vitamin B₆) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.     

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O(2)(-)) and nitric oxide (NO) forms peroxynitrite (ONOO(-)) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO(-). Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O(2)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO(-). We also found that ONOO(-) directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO(-) formed oligomers. Resveratrol (RES), a scavenger of O(2)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) and ONOO(-) levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO(-) during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.     

Purification and characterization of farnesyl pyrophosphate synthase from Capsicum annuum

Farnesyl pyrophosphate synthase (FPP) displaying dimethylallyl transferase activity (EC 2.5.1.1) and geranyl transferase activity (EC 2.5.1.10) was purified from Capsicum fruits. This prenyltransferase has a molecular mass of 89,000 +/- 5000 Da resulting from the association of two apparently identical subunits having a molecular mass of 43,000 +/- 2000 Da. Antibodies raised against Capsicum FPP synthase selectively blocked the transferase activity. Analysis of the immunological relationships between FPP synthase and geranylgeranyl pyrophosphate synthase (EC 2.5.1.1, EC 2.5.1.10 and EC 2.5.1.30) revealed that these two enzymes though performing the same mechanism of catalysis and accepting identical substrates have different antigenic determinants. Thus, in connection to previous work, this immunological study suggests that Capsicum FPP is strictly located in the extraplastidial compartment.     

Developmental and stress regulation of gene expression for plastid and cytosolic isoprenoid pathways in pepper fruits.

Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals.     

Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2(-*)), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2(-*), or an antagonist of the BK(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2(-*) generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.     

Inhibition of lycopene cyclization by Capsicum chromoplast membranes by 2-aza-2,3-dihydrosqualene

2-Aza-2,3-dihydrosqualene strongly inhibited lycopene cyclase from Capsicum chromoplast membranes.     

Identification of a novel series of benzimidazoles as potent and selective antagonists of the human melanocortin-4 receptor

A novel series of benzimidazoles was identified and optimized, leading to the discovery of potent and selective antagonists of the human melanocortin-4 receptor. In addition, compound 5i was shown to cross the blood-brain barrier after intravenous dosing in rats.     

Candidate loci reveal genetic differentiation between temporally divergent migratory runs of Chinook salmon (Oncorhynchus tshawytscha)

Local adaptation is a dynamic process driven by selection that can vary both in space and time. One important temporal adaptation for migratory animals is the time at which individuals return to breeding sites. Chinook salmon (Oncorhynchus tshawytscha) are excellent subjects for studying the genetic basis of temporal adaptation because their high seasonal homing fidelity promotes reproductive isolation leading to the formation of local populations across diverse environments. We tested for adaptive genetic differentiation between seasonal runs of Chinook salmon using two candidate loci; the circadian rhythm gene, OtsClock1b, and Ots515NWFSC, a microsatellite locus showing sequence identity to three salmonid genes central to reproductive development. We found significant evidence for two genetically distinct migratory runs in the Feather River, California (OtsClock1b: F(ST)=0.042, P=0.02; Ots515NWFSC: F(ST)=0.058, P=0.003). In contrast, the fall and threatened spring runs are genetically homogenous based on neutral microsatellite data (F(ST)=-0.0002). Similarly, two temporally divergent migratory runs of Chinook salmon from New Zealand are genetically differentiated based on polymorphisms in the candidate loci (OtsClock1b: F(ST)=0.083, P-value=0.001; Ots515NWFSC: F(ST)=0.095, P-value=0.000). We used an individual-based assignment method to confirm that these recently diverged populations originated from a single source in California. Tests for selective neutrality indicate that OtsClock1b and Ots515NWFSC exhibit substantial departures from neutral expectations in both systems. The large F(ST )estimates could therefore be the result of directional selection. Evidence presented here suggests that OtsClock1b and Ots515NWFSC may influence migration and spawning timing of Chinook salmon in these river systems.