Redox studies of subunit interactivity in aerobic ribonucleotide reductase from Escherichia coli

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(2)) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (-163 +/- 3 mV) different from that of R2(Met) produced by hydroxyurea (-115 +/- 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming -223 +/- 6 and -226 +/- 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to -192 +/- 2 mV for dGDP/dTTP and to -203 +/- 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mm) and dATP (0.3-0.6 mm) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.     

Bifunctional agents for reperfusion arrhythmias: novel hybrid vitamin E/class I antiarrhythmics

We have synthesized a series of hybrid compounds combining the pharmacophoric redox moieties of vitamin E and key features responsible for the antiarrhythmic properties of the class I antiarrhythmics procainamide and lidocaine. Procainamide analogue (2a) and lidocaine analogues (14a) and (14b) are very strong inhibitors of lipid peroxidation. All analogues tested at 100 or 30 microM enhanced the post ischemic recovery without inducing ventricular fibrillations while there was no evidence in our experiments for drug-induced pro-arrhythmia. In addition, they induced a widening of the QRS intervals. Our data suggest that the efficacy of the new compounds in preventing reperfusion arrhythmias could be attributed to their combined effects involving inhibition of free radical mediated damage coupled with antiarrhythmic properties.     

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes.     

Ion channels are linked to differentiation in keratinocytes

Summary In vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca²⁺. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca²⁺, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca²⁺ and activated by physiological concentrations of Ca²⁺. We also found a 35 pS Cl^– channel whose open probability increased with depolarization. Finally, a 70 pS K⁺ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl^– channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K⁺ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.This work was supported in part by a National Institutes of Health grant K08 AR01853-03 and a National Science Foundation grant DCB-9009915 (to T.M.M.); National Institutes of Health Research Career Development Award K04 ARO 1803 and AR 39031 (to R.R.I.) and a National Institutes of Health grant GM-44840 (to P.A.P.).     

Distribution of methionine- and leucine-enkephalin within the rat pituitary gland measured by highly specific radioimmunoassays

The distribution of methionine- and leucine-enkephalin within the rat pituitary gland was measured using highly specific antisera in conjunction with purification by high performance liquid chromatography. The highest concentrations were found in the pars intermedia (7 pmole/mg for methionine-enkephalin, 4 pmole/mg for leucine-enkephalin), whilst the pars nervosa contained 2.2 pmole/mg of each and the pars anterior the least (methionine-enkephalin: 0.51 pmole/mg, leucine-enkephalin: 0.36 pmole/mg).     

Recombination Marks the Evolutionary Dynamics of a Recently Endogenized Retrovirus

All vertebrate genomes have been colonized by retroviruses along their evolutionary trajectory. Although endogenous retroviruses (ERVs) can contribute important physiological functions to contemporary hosts, such benefits are attributed to long-term coevolution of ERV and host because germline infections are rare and expansion is slow, and because the host effectively silences them. The genomes of several outbred species including mule deer (Odocoileus hemionus) are currently being colonized by ERVs, which provides an opportunity to study ERV dynamics at a time when few are fixed. We previously established the locus-specific distribution of cervid ERV (CrERV) in populations of mule deer. In this study, we determine the molecular evolutionary processes acting on CrERV at each locus in the context of phylogenetic origin, genome location, and population prevalence. A mule deer genome was de novo assembled from short- and long-insert mate pair reads and CrERV sequence generated at each locus. We report that CrERV composition and diversity have recently measurably increased by horizontal acquisition of a new retrovirus lineage. This new lineage has further expanded CrERV burden and CrERV genomic diversity by activating and recombining with existing CrERV. Resulting interlineage recombinants then endogenize and subsequently expand. CrERV loci are significantly closer to genes than expected if integration were random and gene proximity might explain the recent expansion of one recombinant CrERV lineage. Thus, in mule deer, retroviral colonization is a dynamic period in the molecular evolution of CrERV that also provides a burst of genomic diversity to the host population.     

The genetic population structure and temporal genetic stability of gilthead sea bream Sparus aurata populations in the Aegean and Ionian Seas,using microsatellite DNA markers

We examined 662 gilthead sea bream Sparus aurata from wild samples of the species in the Aegean and Ionian Seas, using 20 EST-linked microsatellite markers, in three multiplex panels, as well as seven anonymous loci. Most of the markers were revealed to be highly polymorphic. We found low genetic differentiation between the sampling stations/areas with total F_ST 0.002 (P < 0.05). Based on comparison of five temporal samples, our results indicate genetic data consistency over time for all tested samples, pointing to stable populations, despite reported repeated escape events. Our results confirm the genetic population structure previously observed in these specific areas, using by far more markers than in previous studies in both coding and non-coding DNA loci. The limited genetic structure and the temporal genetic stability indicate neither major genetic differentiation of local populations by geographic isolation nor influence from anthropogenic factors. These results provide a baseline for future reference in any management programme of both wild and farmed population of S. aurata as well as of other aquaculture species with a potential introgression among farmed and wild populations.     

Correlation of ovary and leaf spermidine and spermine content with the alternate bearing habit of olive

In order to investigate the possible role of the polyamines spermidine and spermine, in the alternate bearing habit of the olive, a comparative analysis of their content in vegetative and reproductive organs during the flowering period of the 'on' and 'off' years, was conducted. Samples of flowers, ovaries and leaves from two cultivars, the biennial bearer 'Chondrolia Chalkidikis' and the regular cropping 'Koroneiki' were used and the free and conjugated forms of polyamines were detected. In addition, the concentrations of K(+) and Ca(2+) were determined in all samples. Spermidine conjugates were predominant in all samples, while free spermine was not detectable. Conjugated forms of both spermidine and spermine were accumulated in ovaries, rather than in other floral tissues (corolla and stamens). They also, accumulated in significantly higher concentrations in ovaries and leaves of both cultivars, during the 'on' compared with the 'off' year, and in much higher concentrations, during the 'on' year, in ovaries of the strong alternate bearer 'Chondrolia Chalkidikis' than in 'Koroneiki'. Free spermidine was detected in ovaries and leaves of both cultivars, showing a decline during ovule fertilization in both years. Such a decline was not always recorded for the conjugated forms, depending on the organ studied and the flower load. A relation between endogenous spermidine and spermine with the K(+) or Ca(2+) composition was not established.