Capture of MicroRNA–Bound mRNAs Identifies the Tumor Suppressor miR-34a as a Regulator of Growth Factor Signaling

A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division

Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

BACKGROUND: Inositol 1,4,5trisphosphate (IP(3)) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab)) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab) alleles are phenotypically normal. However, the presence of one Plcd3(mNab) allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3(mNab) mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab) mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface

Signal processing and transduction in plant cells: the end of the beginning?

Plants have a very different lifestyle to animals, and one might expect that unique molecules and processes would underpin plant-cell signal transduction. But, with a few notable exceptions, the list is remarkably familiar and could have been constructed from animal studies. Wherein, then, does lifestyle specificity emerge

Dental enamel cells express functional SOCE channels

Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation

HPV Vaccine Acceptance among Latina Mothers by HPV Status

Objective: We investigated whether Latina mothers who were and were not human papillomavirus (HPV) positive differed in their knowledge and acceptance of the HPV vaccine for their children. Methods: We conducted a cross–sectional survey among women aged 18–64 years between April 2007 and April 2008. Data collectors conducted in-person interviews in community clinics with 215 HPV-negative women and 190 HPV-positive women (with respective response rates of 64% and 84%). Most (83%) HPV-positive women were recruited at dysplasia clinics. Although no HPV-negative women were recruited at dysplasia clinics, they were recruited at other low-income public and private clinics. Results: After adjustment for age, marital status, and health insurance, women who were HPV positive were more likely than HPV-negative women to have heard about the HPV vaccine, to indicate they would have their daughters and sons vaccinated against cervical cancer even if they had to pay themselves, and to be in favor of the proposed Texas law requiring girls to receive the HPV vaccine before entry into sixth grade but less likely to be in favor of girls receiving the vaccine at age _13. Conclusions: Our findings indicate that \u3e90% of Latinas living on the Texas-Mexico border find the HPV vaccine acceptable for their own daughters and sons

Physiological impacts of elevated carbon dioxide and ocean acidification on fish

Most fish studied to date efficiently compensate for a hypercapnic acid-base disturbance; however, many recent studies examining the effects of ocean acidification on fish have documented impacts at CO2 levels predicted to occur before the end of this century. Notable impacts on neurosensory and behavioral endpoints, otolith growth, mitochondrial function, and metabolic rate demonstrate an unexpected sensitivity to current-day and near-future CO2 levels. Most explanations for these effects seem to center on increases in Pco2 and HCO3- that occur in the body during pH compensation for acid-base balance; however, few studies have measured these parameters at environmentally relevant CO2 levels or directly related them to reported negative endpoints. This compensatory response is well documented, but noted variation in dynamic regulation of acid-base transport pathways across species, exposure levels, and exposure duration suggests that multiple strategies may be utilized to cope with hypercapnia. Understanding this regulation and changes in ion gradients in extracellular and intracellular compartments during CO2 exposure could provide a basis for predicting sensitivity and explaining interspecies variation. Based on analysis of the existing literature, the present review presents a clear message that ocean acidification may cause significant effects on fish across multiple physiological systems, suggesting that pH compensation does not necessarily confer tolerance as downstream consequences and tradeoffs occur. It remains difficult to assess if acclimation responses during abrupt CO2 exposures will translate to fitness impacts over longer timescales. Nonetheless, identifying mechanisms and processes that may be subject to selective pressure could be one of many important components of assessing adaptive capacity