Pyramid Screening: Combining Three Genetic Screens into One Efficient Screen for Shoot Regeneration Mutants in Arabidopsis thaliana

Gain-of-function genetic mutants are typically found by creating a non-permissive condition and screening for plants that overcome the stress. Separate genetic screens are conducted for each condition, a potentially time-consuming effort. In severed Arabidopsis thaliana leaves, high light, suboptimal hormone exposure and old age were each independently found to reduce frequency of shoot regeneration. Rather than conducting three separate mutant screens to dissect these three pathways, a laborious process, we hypothesized that we could undertake a single economical screen to retrieve mutations specific for each trait as well as cross-talk alleles between pathways. Instead of creating non-permissive conditions for each of our three traits of interest, we combined the three suboptimal stress conditions such that only when combined was shoot regeneration abolished. No one stress was primarily responsible for loss of our trait, thus ensuring that we could recover mutant alleles in any of the three pathways of interest. Screening of 18,000 mutagenized plants resulted in 12 SHOOTING UP (stu) mutants. Secondary screening revealed that we had recovered alleles that were both specific for a pathway (light, hormones or age) and which acted through multiple pathways. Our approach, which we refer to as pyramid screening, represents an economical method for mutant screening of multiple pathways in parallel (three screens in one) and has the potential to recover alleles that cross-talk between multiple pathways that underlie a complex trait such as organ regeneration. Pyramid screening should be widely applicable across species.

     

Direct Pro-Inflammatory Effects of Prorenin on Microglia

Neuroinflammation has been implicated in hypertension, and microglia have been proposed to play an important role in the progression of this disease. Here, we have studied whether microglia are activated within cardiovascular regulatory area(s) of the brain during hypertension, especially in high blood pressure that is associated with chronic activation of the renin-angiotensin-system. In addition, we determined whether prorenin, an essential component of the renin-angiotensin-system, exerts direct pro-inflammatory effects on these microglia. Our data indicate that two rodent models which display neurogenic hypertension and over activation of the renin-angiotensin-system in the brain (sRA mice and spontaneously hypertensive rats) exhibit microglial activation, and increased levels of pro-inflammatory cytokines, in the paraventricular nucleus of the hypothalamus, an area crucial for regulation of sympathetic outflow. Further, the renin-angiotensin-system component prorenin elicits direct activation of hypothalamic microglia in culture and induction of pro-inflammatory mechanisms in these cells, effects that involve prorenin receptor-induced NFκB activation. In addition, the prorenin-elicited increases in cytokine expression were fully abolished by microglial inhibitor minocycline, and were potentiated by pre-treatment of cells with angiotensin II. Taken together with our previous data which indicate that pro-inflammatory processes in the paraventricular nucleus are involved in the hypertensive action of renin-angiotensin-system, the novel discovery that prorenin exerts direct stimulatory effects on microglial activation and pro-inflammatory cytokine production provides support for the idea that renin-angiotensin-system -induced neurogenic hypertension is not restricted to actions of angiotensin II alone.     

Angiotensin II type 1 receptor-modulated signaling pathways in neurons

Mammalian brain contains high densities of angiotensin II (Ang II) type 1 (AT1) receptors, localized mainly to specific nuclei within the hypothalamus and brainstem regions. Neuronal AT1 receptors within these areas mediate the stimulatory actions of central Ang II on blood pressure, water and sodium intake, and vasopressin secretion, effects that involve the modulation of brain noradrenergic pathways. This review focuses on the intracellular events that mediate the functional effects of Ang II in neurons, via AT1 receptors. The signaling pathways involved in short-term changes in neuronal activity, membrane ionic currents, norepinephrine (NE) release, and longer-term neuromodulatory actions of Ang II are discussed. It will be apparent from this discussion that the signaling pathways involved in these events are often distinct.     

Inhibition of miR-144/199 promote myeloma pathogenesis via upregulation of versican and FAK/STAT3 signaling

Molecular and Cellular Biochemistry - The continuous rise in relapse rate and mortality for multiple myeloma (MM) demands an effective treatment option. The microRNAs are emerging nowadays for...     

Insulin Stimulates Phosphatidylinositol 3-Kinase Activity in Rat Neuronal Primary Cultures

Abstract: The effect of cortical spreading depression (CSD) on cerebral protein synthesis (CPS) was examined. CSD was evoked in normal rats with KCI, and CPS was measured autoradiographically with [1-¹⁴C]leucine. Average rates (mean ± SD) of CPS in layers I-IV of cortex decreased significantly from 10.7 ± 0.6 (sham-operated controls; n = 4) to 6.7 ± 0.7 nmol/g/min (n = 4; p < 0.01) and in layers V-VI from 10.9 ± 0.5 to 9.4 ± 0.4 nmol/g/min (p < 0.05) during 60 min of repetitive CSD. Spreading depression did not affect CPS rates in other subcortical brain regions. These results indicate that KCl-evoked CSD induces inhibition but not suppression of cortical protein synthesis.     

Toxicological evaluation of karaya gum; acute and subacute oral toxicity in rats

Male and female albino rats (Wistar strain) were given single and multiple doses of karaya gum suspended either in peanut oil or mixed with basal diet at different concentrations ranging from 0.5 to 8 g gum/kg body weight. The plant gum did not elicit any overt signs of toxicity or death in both sexes of rats. Daily administration of karaya gum mixed with basal diet at different dose levels (0, 5, 20 and 40 g gum/kg diet) for a period of 90 days showed no adverse effects in male and female rats. The body weight, growth pattern, food and water intake were comparable with those of the normal rats. There were no significant biochemical, or morphological alterations in the vital organs of experimental animals.     

Regulation of Angiotensin II–induced Neuromodulation by MARCKS in Brain Neurons

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A–sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg²⁺ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg²⁺ sensitive but cyclosporin insensitive.     

Ontogeny of insulin receptors in the rat hemochorial placenta

Binding of 125I-insulin to rat placental membranes was time and protein concentration dependent, reversible, and specific. Unlabeled porcine insulin competed for 125I-insulin binding with an IC50 of 65 nM, while IGF-I was much less potent with an IC50 of 2.12 mM. Specific binding of 125I-insulin decreased during the second half of gestation from Days 11 to 19. Scatchard analysis of the binding data for membranes prepared from Gestation Days 11 and 19 yielded typical curvilinear plots which showed a marked decrease in the number of binding sites in late gestation placenta. Beginning on Day 14, insulin binding was characterized with isolated labyrinth and basal zone portions of the hemochorial placenta. There was no evidence for differences in Kd values or the number of binding sites in these two functionally distinct portions of the rat placenta. Crosslinking of 125I-insulin followed by SDS-PAGE showed a single protein with a molecular weight of 130,000 from placental tissues on Gestation Days 11 and 19 and confirmed a gestational decrease in the number of insulin receptors. In solubilized, lectin-purified preparations from placenta and liver membranes, insulin stimulated the phosphorylation of a Mr 95,000 protein. 32P-incorporation into this 95,000 protein was stimulated fivefold by insulin in Day 11 placenta receptor, whereas no detectable 32P-incorporation was found in Day 19 placenta. Thus, while the alpha- and beta-subunits of insulin receptors in mid and late gestation placenta have molecular weights which are similar to receptors in maternal liver, data indicate the presence of a functional difference in insulin-stimulated kinase activities.