Characteristics of brain Kv1 channels tailored to mimic native counterparts by tandem linkage of alpha subunits: implications for K+ channelopathies

Most neuronal Kv1 channels contain Kv1.1, Kv1.2 alpha, and Kvbeta2.1 subunits, yet the influences of their stoichiometries on properties of the (alpha)(4)(beta)(4) variants remain undefined. cDNAs were engineered to contain 0, 1, 2, or 4 copies of Kv1.1 with the requisite number of Kv1.2 and co-expressed in mammalian cells with Kvbeta2.1 to achieve "native-like" hetero-oligomers. The monomeric (Kv1.1 or 1.2), dimeric (Kv1.1-1.2 or 1.2-1.2), and tetrameric (Kv1.1-(1.2)(3)) constructs produced proteins of M(r) approximately 62,000, 120,000, and 240,000, which assembled into (alpha)(4)(beta)(4) complexes. Each alpha cRNA yielded a distinct K(+) current in oocytes, with voltage dependence of activation being shifted negatively as the Kv1.1 content in tetramers was increased. Channels containing 1, 2, or 4 copies of Kv1.1 were blocked by dendrotoxin k (DTX)(k) with similarly high potencies, whereas Kv(1.2)(4) proved nonsusceptible. Accordingly, Kv1.2/beta2.1 expressed in baby hamster kidney cells failed to bind DTX(k); in contrast, oligomers containing only one Kv1.1 subunit in a tetramer exhibited high affinity, with additional copies causing modest increases. Thus, one Kv1.1 subunit largely confers high affinity for DTX(k), whereas channel electrophysiological properties are tailored by the content of Kv1.1 relative to Kv1.2. This notable advance could explain the diversity of symptoms of human episodic ataxia I, which is often accompanied by myokymia, due to mutated Kv1.1 being assembled in different combinations with wild-type and Kv1.2.     

RGS9-2 is a negative modulator of mu-opioid receptor function

Regulators of G-protein signaling (RGS) 9-2 is a striatal enriched protein that controls G protein coupled receptor signaling duration by accelerating Galpha subunit guanosine triphosphate hydrolysis. We have previously demonstrated that mice lacking the RGS9 gene show enhanced morphine analgesia and delayed development of tolerance. Here we extend these studies to understand the mechanism via which RGS9-2 modulates opiate actions. Our data suggest that RGS9-2 prevents several events triggered by mu-opioid receptor (MOR) activation. In transiently transfected PC12 cells, RGS9-2 delays agonist induced internalization of epitope HA-tagged mu-opioid receptor. This action of RGS9-2 requires localization of the protein near the cell membrane. Co-immunoprecipitation studies reveal that RGS9-2 interacts with HA-tagged mu-opioid receptor, and that this interaction is enhanced by morphine treatment. In addition, morphine promotes the association of RGS9-2 with another essential component of MOR desensitization, beta-arrestin-2. We also show that over-expression of RGS9-2 prevents opiate-induced extracellular signal-regulated kinase phosphorylation. Our data indicate that RGS9-2 plays an essential role in opiate actions, by negatively modulating MOR downstream signaling as well as the rate of MOR endocytosis.     

Combining immunological and molecular data to assess phylogenetic relations of some Greek Podarcis species

Most recent molecular studies revealed the phylogeny of Greek Podarcis species, which for years remained elusive, due to discordant data produced from various chromosomal, complement fixation and protein studies. In this report, we analyzed cellular immune responses of spleen-derived lymphocytes from six allopatric Podarcis species encountered in Greece, by assessing two-way mixed lymphocyte reaction (MLR)-induced proliferation. On the basis of stimulation indices (S.I.) as determined from cultures set up from xenogeneic splenocytes coincubated in pairs, we generated a phylogenetic tree, fully consistent with the phylogenetic relationships of Podarcis as determined by parallel analyses based on partial mitochondrial (mt) DNA sequences. Although the exact mechanisms triggering lymphocyte responses in lizard two-way xenogeneic MLR are not fully understood, our results show the potential use of cell-mediated immune responses as an additional approach to mtDNA analysis, for species delimitation within specific lizard taxa.     

Differentiation-induced alterations in cyclic AMP signaling in the Cath.a differentiated (CAD) neuronal cell line

Regulation of intracellular cyclic AMP is critical to the modulation of many cellular activities, including cellular differentiation. Moreover, morphological differentiation has been linked to subsequent alterations in the cAMP signaling pathway in various cellular models. The current study was designed to explore the mechanism for the previously reported enhancement of adenylate cyclase activity in Cath.a differentiated cells following differentiation. Differentiation of Cath.a differentiated cells stably expressing the D2L dopamine receptor markedly potentiated both forskolin- and A2-adenosine receptor-stimulated cAMP accumulation. This enhancement was accompanied by a twofold increase in adenylate cyclase 6 (AC6) expression and a dramatic loss in the expression of AC9. The ability of Ca2+ to inhibit drug-stimulated cAMP accumulation was enhanced following differentiation, as was D2L dopamine receptor-mediated inhibition of Galphas-stimulated cAMP accumulation. Differentiation altered basal and drug-stimulated phosphorylation of the cAMP-response element-binding protein, which was independent of changes in protein kinase A expression. The current data suggest that differentiation of the neuronal cell model, Cath.a differentiated cells induces significant alterations in the expression and function of both the proximal and distal portions of the cAMP signaling pathway and may impact cellular operations dependent upon this pathway.     

Switching between forest and trees: Opposite relationship of progesterone and testosterone to global–local processing

Sex differences in attentional selection of global and local components of stimuli have been hypothesized to underlie sex differences in cognitive strategy choice. A Navon figure paradigm was employed in 32 men, 41 naturally cycling women (22 follicular, 19 luteal) and 19 users of oral contraceptives (OCs) containing first to third generation progestins in their active pill phase. Participants were first asked to detect targets at any level (divided attention) and then at either the global or the local level only (focused attention). In the focused attention condition, luteal women showed reduced global advantage (i.e. faster responses to global vs. local targets) compared to men, follicular women and OC users. Accordingly, global advantage during the focused attention condition related significantly positively to testosterone levels and significantly negatively to progesterone, but not estradiol levels in a multiple regression model including all naturally cycling women and men. Interference (i.e. delayed rejection of stimuli displaying targets at the non-attended level) was significantly enhanced in OC users as compared to naturally cycling women and related positively to testosterone levels in all naturally cycling women and men. Remarkably, when analyzed separately for each group, the relationship of testosterone to global advantage and interference was reversed in women during their luteal phase as opposed to men and women during their follicular phase. As global processing is lateralized to the right and local processing to the left hemisphere, we speculate that these effects stem from a testosterone-mediated enhancement of right-hemisphere functioning as well as progesterone-mediated inter-hemispheric decoupling.     

Egg laying preference of Sesamia nonagrioides (Lepidoptera: Noctuidae) among primary and secondary hosts

Sesamia nonagrioides Lefébvre (Lepidoptera: Noctuidae) has a fairly wide range of host plants. The present study tests the egg-laying preference of S. nonagrioides on four potential host plants (maize, sweet and fiber sorghum and johnsongrass) with respect to several life history traits. No-choice and two-choice tests in all possible combinations were conducted. Concerning no-choice tests, significantly higher number of eggs were laid on sweet sorghum (282.5 eggs/female) in relation to the other tested plants. Moreover, viability of eggs that were laid on sweet sorghum was significantly higher in relation to those that were laid on johnsongrass. Longevity of females reared on maize plants was significantly greater compared with the other test plants except johnsongrass. Furthermore, pre-oviposition period of females reared on maize was significantly longer (2.4-fold) compared with that on sweet sorghum and johnsongrass. Similarly post-oviposition period of females reared on maize was significantly longer compared with that on fiber sorghum. On two choice tests significantly fewer eggs were laid on sweet sorghum than on maize. On the other hand, significantly more eggs were laid on sweet sorghum than on fiber sorghum and johnsongrass. We discuss the potential of secondary hosts to be used as trap plants.     

Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P?=?.002, P?<?.0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P?=?.04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5?months, P?=?.03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P?=?.03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.