Intermediate Ca2+-Sensitive K+ Channels are Necessary for Prolactin-Induced Proliferation in Breast Cancer Cells

Prolactin (PRL) is a polypeptidic hormone which acts both systemically and locally to cause lactation by interacting with the PRL receptor, a Janus kinase (JAK2)-coupled cytokine receptor family member. Several studies have reported that serum PRL level elevation is associated with an increased risk for breast cancer, and evidence has suggested that PRL is one actor in the pathogenesis and progression of this cancer. We previously reported the involvement of hIKCa1 in breast cell cycle progression and cell proliferation. However, mechanisms by which PRL cooperates with these channels to modulate breast epithelial cell proliferation remain unknown. Our results showed that, in the MCF-7 breast cancer cell line, PRL increased hIKCa1 current density. These channels were functional and regulated the resting membrane potential. The PRL effects were inhibited by TRAM-34 and clotrimazole, the most used hIKCa1 blockers. Moreover, PRL increased proliferation in a dose-dependent manner without overexpressing hIKCa1. To determine whether PRL-induced proliferation and hIKCa1 activity involved the JAK2 pathway, we used pharmacological JAK2 inhibitors (AG490 and JAK inhibitor I). Indeed, PRL-induced JAK2 phosphorylation was required for both cell proliferation and hIKCa1 activity. In the presence of either hIKCa1 blockers or siRNA-hIKCa1, PRL failed to increase cell proliferation and hIKCa1 activity. Taken together, our results demonstrate that PRL plays a role in breast cancer cell proliferation by increasing hIKCa1 activity through the JAK2 signaling pathway.     

Internet-Based Brief Intervention to Prevent Unhealthy Alcohol Use among Young Men: A Randomized Controlled Trial

Introduction

Alcohol use is one of the leading modifiable morbidity and mortality risk factors among young adults.

Study Design

2 parallel-group randomized controlled trial with follow-up at 1 and 6 months.

Setting/Participants

Internet based study in a general population sample of young men with low-risk drinking, recruited between June 2012 and February 2013.Intervention: Internet-based brief alcohol primary prevention intervention (IBI). The IBI aims at preventing an increase in alcohol use: it consists of normative feedback, feedback on consequences, calorific value alcohol, computed blood alcohol concentration, indication that the reported alcohol use is associated with no or limited risks for health. Intervention group participants received the IBI. Control group (CG) participants completed only an assessment.

Main Outcome Measures

Alcohol use (number of drinks per week), binge drinking prevalence. Analyses were conducted in 2014–2015.

Results

Of 4365 men invited to participate, 1633 did so; 896 reported low-risk drinking and were randomized (IBI: n = 451; CG: n = 445). At baseline, 1 and 6 months, the mean (SD) number of drinks/week was 2.4(2.2), 2.3(2.6), 2.5(3.0) for IBI, and 2.4(2.3), 2.8(3.7), 2.7(3.9) for CG. Binge drinking, absent at baseline, was reported by 14.4% (IBI) and 19.0% (CG) at 1 month and by 13.3% (IBI) and 13.0% (CG) at 6 months. At 1 month, beneficial intervention effects were observed on the number of drinks/week (p = 0.05). No significant differences were observed at 6 months.

Conclusion

We found protective short term effects of a primary prevention IBI.

Trial Registration

Controlled-Trials.com ISRCTN55991918     

Down‐regulation of Orai3 arrests cell‐cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells

Breast cancer (BC) is the leading cancer in the world in terms of incidence and mortality in women. However, the mechanism by which BC develops remains largely unknown. The increase in cytosolic free Ca²⁺ can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca²⁺ selective channels. In the present study, we analyzed Orai3 expression in normal and cancerous breast tissue samples, and its role in MCF‐7 BC and normal MCF‐10A mammary epithelial cell lines. We found that the expression of Orai3 mRNAs was higher in BC tissues and MCF‐7 cells than in normal tissues and MCF‐10A cells. Down‐regulation of Orai3 by siRNA inhibited MCF‐7 cell proliferation and arrested cell cycle at G1 phase. This phenomenon is associated with a reduction in CDKs 4/2 (cyclin‐dependent kinases) and cyclins E and D1 expression and an accumulation of p21^Waf1/Cip1 (a cyclin‐dependent kinase inhibitor) and p53 (a tumor‐suppressing protein). Orai3 was also involved in MCF‐7 cell survival. Furthermore, Orai3 mediated Ca²⁺ entry and contributed to intracellular calcium concentration ([Ca²⁺]_i). In MCF‐10A cells, silencing Orai3 failed to modify [Ca²⁺]_i, cell proliferation, cell‐cycle progression, cyclins (D1, E), CDKs (4, 2), and p21^Waf1/Cip1 expression. Our results provide strong evidence for a significant effect of Orai3 on BC cell growth in vitro and show that this effect is associated with the induction of cell cycle and apoptosis resistance. Our study highlights a possible role of Orai3 as therapeutic target in BC therapy. J. Cell. Physiol. 226: 542–551, 2011. © 2010 Wiley‐Liss, Inc.     

Variations in the vulvar temperature of sows during proestrus and estrus as determined by infrared thermography and its relation to ovulation

The prediction of ovulation time is one of the most important and yet difficult processes in pig production, and it has a considerable impact on the fertility of the herd and litter size. The objective of this study was to assess the vulvar skin temperature of sows during proestrus and estrus using infrared thermography and to establish a possible relationship between the variations in vulvar temperature and ovulation. The experimental group comprised 36 crossbred Large White × Landrace females, of which 6 were gilts and 30 were multiparous sows. Estrus was detected twice daily and the temperature was obtained every 6 hours from the vulvar area and from two control points in the gluteal area (Gluteal skin temperature [GST]). A third variable, vulvar–gluteal temperature (VGT) was obtained from the difference between the vulvar skin temperature and the GST values. The animals were divided into two subgroups: group A consisting of 11 animals with estrus detected at 6:00 AM, Day 4 postweaning, and group B comprising seven animals with estrus detected at 6:00 AM, Day 5 post-weaning. Both groups showed a similar trend in the VGT. The VGT increased during the proestrus, reaching a peak 24 hours before estrus in group A and 48 hours before estrus in group B. The VGT then decreased markedly reaching the lowest value in groups A and B, respectively, 12 and 6 hours after estrus. Although the time of ovulation was only estimated on the basis of a literature review, the matching between the temporal variations of the VGT values and the predicted time of the peak of estradiol secretion that ultimately leads to the ovulation processes suggests that the VGT values represent a potential predictive marker of the ovulatory events.     

Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

Background

Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.

Objective

The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.

Methods

Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.

Results

Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.

Conclusions & Clinical Relevance

DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.     

Improving performance of olive trees by the enhancement of key physiological parameters of olive leaves in response to foliar fertilization

Most of the studies investigated the effects of nutrient-based fertilizers on olive fruits and oil quality; few studies have been interested in the modification of the chemical composition of olive leaves in response to fertilization. Thus, the current study aims to examine the effects of foliar fertilization on the mineral profile of olive leaves as well as the concentrations of chlorophyll, antioxidants (phenolic compounds) and carbohydrates. Experimentation consists of the annual application of six treatments during two successive growing seasons (2009–2010): TC (untreated trees), P1 (nitrogen-based fertilizer), P2 (contains boron, magnesium and manganese), P3 (phosphorus and potassium based fertilizer), P4 (rich in calcium and phosphorus), T5 (P1 and P2 application) and T6 (P1, P2, P3 and P4 application). At the end of the experiment, mineral analysis of olive leaves showed an increase in the concentrations of most nutrients which induced changes in biochemical composition: an increase of chlorophyll content, a reduction of total phenols and oleuropein concentrations coupled with an increase of hydroxytyrosol level. Moreover, an increase of total sugar content and most individual sugars, principally translocated forms of sugars (mannitol, sucrose and raffinose), was also observed. The accumulation of these key physiological parameters by foliar fertilization suggests an improvement of physiological performance and photosynthetic capacity of olive trees. Moreover, from a biological point of view, the results of the study revealed the possibility to improve plants of medicinal interest by enhancing the accumulation of some bio-active compounds, such as hydroxytyrosol and mannitol, via foliar nutrient supply.     

Hormonal status of cortisol and dehydroepiandrosterone sulfate in an elderly Tunisian population

Adrenal function and aging have been the object of intense interest recently, especially as regards dehydroepiandrosterone sulfate (DHEA-S), which is of major importance, since it is distinct from cortisol and aldosterone in declining with age. In a group of healthy old Tunisians, we investigated the association between cortisol and DHEA-S, on the one hand, and age, sex, lifestyle, physical health, including the body mass index (BMI), physical activity, and smoking indicators, on the other hand. We observed that cortisol concentrations did not change with aging, while DHEA-S concentrations decrease with age in both sexes. Cortisol/DHEA-S ratio, however, increases with aging. Our results revealed that DHEA-S levels are affected neither by physical activity nor by weight. It appears also that current smoking could not affect the level of DHEA-S. Relationships were found between DHEA-S concentrations and BMI, then between DHEA-S levels and serum cholesterol, triglycerides and calcium. No modification in the morning serum cortisol was found to be associated with aging. Decrease in DHEA-S levels is, however, clearly associated with this phenomenon. High cortisol/DHEA-S ratio accelerates the occurrence of some adult diseases, such as diabetes mellitus, atherosclerosis, dementia, and osteoporosis. Generally, the adrenal insufficiency marked by a cognitive impairment, immune disorders, sexual dysfunction, and scores for depression and anxiety can be corrected by a replacement of deficient DHEA-S.     

Immunophenotyping of hematopoietic progenitor cells: Comparison between cord blood and adult mobilized blood grafts

AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 908) using flow cytometry analysis.METHODS: First, we performed a qualitative analysis of CD34+ cell sub-populations in both CB and PAPB grafts using the standardized ISHAGE protocol and a wide panel of 20 monoclonal antibodies. Next, we stud-ied some parameters, such as the age of mothers and the weight of newborns, which can influence the qual-ity and the quantity of CD34+ cells from CB. RESULTS: We found that the percentage of apoptotic cells was high in CB in comparison to PAPB (PAPB: 4.6% ± 2.6% vs CB: 53.4% ± 5.2%, P < 0.001). In CB, the weight of newborn and the age of the mother have the influence on CD34+ cells. The follow-up of Ag CD133in the ISHAGE double platform protocol in association with CD45, CD34 and the 7’AAD shows an equal rate between the two cell populations CD133+CD45+CD34+ high and CD34+CD45+ high with a higher percentage. So, is the inclusion of Ac CD133 necessary in the pres-ent panel included in the ISHAGE methodflLast part, we showed a signif icant presence of interferon γ in CB in comparison to PAPB, the annexin showing the high number of apoptotic cells in CB. CONCLUSION: This study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking umbilical CB units for transplantation.     

<Emphasis Type="Italic">Brassica napus</Emphasis> possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> infection,wounding and defense hormone treatment

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form beta-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.