HPLC analysis of estrogen receptor by a multidimensional approach

Previously we demonstrated the polymorphism of estrogen receptors (ER) in cytosol of various tissues based upon properties of size, shape and surface charge. This study describes the application of a multidimensional approach utilizing HPLC for characterization of ER. Cytosols from human uterus and endometrial carcinomas were characterized sequentially by high performance size exclusion chromatography (HPSEC) on Spherogel TSK-3000 SW, and high performance ion-exchange chromatography (HPIEC) using SynChropak AX-1000 anion exchange columns. Using HPSEC, specific estrogen binding was exhibited by a 30 A isoform and by one appearing after the V0 (approximately 60 A) in human uterus. However, in endometrial carcinoma other smaller binding components with Stoke's radii of less than 20 A were observed also. In buffers containing 400 mM KCl, predominantly a 28-30 A species was observed by HPSEC. Further characterization of the 28-30 A isoform from low and high salt elution from HPSEC was accomplished with an AX-1000 column. With either condition, 2 forms were eluted on HPIEC, 1 in the column wash (retention time 8-9 min), and the other at 50-70 mM phosphate. The elution profile of the larger species (approximately 60 A by HPSEC) on the ion-exchange column was time dependent. Immediate analysis (within 15 min) showed a profile similar to that of the original cytosol which contained minor components eluting in wash buffer and at 50-70 mM phosphate and a major isoform at 180 mM phosphate. However delayed analysis (after 2 h) of the 60 A isoform showed a similar profile (components in buffer wash and at 50-70 mM phosphate) obtained with the 30 A species. This time dependent change was not observed for the 30 A species or for the original cytosol. Estrogen receptors in cytosol sedimented at 10S and 4S in low ionic strength gradients and at 4S in sucrose gradients containing 400 mM KCl. The 28-30 A and 60 A species recovered from HPSEC sedimented at 3.5S. This multidimensional approach indicates that native estrogen receptors dissociated into a number of smaller molecular isoforms, which were distinguishable by different surface charge properties.     

Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support na?ve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.     

Regulation of VEGF in the reproductive tract by sex-steroid hormones

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. In adults, angiogenesis is an infrequent event in the normal tissue except in the female reproductive tract where angiogenesis occurs frequently during the cyclical repair and regeneration of the endometrium as well as in the ovary. Little is known about angiogenesis in the male reproductive tract. The role of VEGF in controlling reproductive tract physiology and the role of hormones in regulating this key regulator of angiogenesis is not well understood. Since reproductive tract physiology is largely under sex-steroid regulation, we have reviewed some recent studies describing the role of sex-steroid hormones in regulating VEGF. We have also included studies on the role of sex-steroids in regulating VEGF and angiogenesis in endometrial, breast and prostate pathologies. We have provided an extensive review of the classical VEGF and VEGF receptors with examples drawn from numerous studies in the literature using diverse biological systems to encourage similar studies in the area of reproductive tract physiology. It is speculated that such studies will provide insights into understanding the role of VEGF in reproductive tract development, causes of infertility, and cancer. Such knowledge would allow us to target VEGF for improving human reproductive tract abnormalities, for enhancing implantation and fertility, and for designing drugs for treatment of endocrine dependent cancers.     

A model for the regulation of cerebral oxygen delivery

On the basis of the assumption that oxygen delivery across theendothelium is proportional to capillary plasmaPO₂, a model is presented that linkscerebral metabolic rate of oxygen utilization(CMR_O2) to cerebral blood flow(CBF) through an effective diffusivity for oxygen (D) of the capillarybed. On the basis of in vivo evidence that the oxygen diffusivityproperties of the capillary bed may be altered by changes in capillaryPO₂, hematocrit, and/or bloodvolume, the model allows changes in D with changes in CBF. Choice inthe model of the appropriate ratio of   (D/D)/(CBF/CBF)determines the dependence of tissue oxygen delivery on perfusion.Buxton and Frank (J. Cereb. Blood Flow. Metab. 17: 64-72, 1997) recently presented alimiting case of the present model in which  = 0. In contrast to thetrends predicted by the model of Buxton and Frank, in the current modelwhen > 0, the proportionality between changes in CBF andCMR_O2 becomes more linear, and similardegrees of proportionality can exist at different basal values ofoxygen extraction fraction. The model is able to fit the observedproportionalities between CBF and CMR_O2 for a large range ofphysiological data. Although the model does not validate any particularobserved proportionality between CBF andCMR_O2, generally values of(CMR_O2/CMR_O2)/(CBF/CBF) close to unity have been observed across ranges of graded anesthesia inrats and humans and for particular functional activations in humans.The model's capacity to fit the wide range of data indicates that theoxygen diffusivity properties of the capillary bed, which can bemodified in relation to perfusion, play an important role in regulatingcerebral oxygen delivery in vivo.

     

Survey of Argentine Health Researchers on the Use of Evidence in Policymaking

Objective

In this study, Argentine health researchers were surveyed regarding their perceptions of facilitators and barriers to evidence-based policymaking in Argentina, as well as their publication activities, and research environment satisfaction.

Methods

A self-administered online survey was sent to health researchers in Argentina. The survey questions were based on a preceding qualitative study of Argentine health researchers, as well as the scientific literature.

Results

Of the 647 researchers that were reached, 226 accessed the survey, for a response rate of 34.9%. Over 80% of researchers surveyed had never been involved in or contributed to decision-making, while over 90% of researchers indicated they would like to be involved in the decision-making process. Decision-maker self-interest was perceived to be the driving factor in the development of health and healthcare policies. Research conducted by a research leader was seen to be the most influential factor in influencing health policy, followed by policy relevance of the research. With respect to their occupational environment, researchers rated highest and most favourably the opportunities available to present, discuss and publish research results and their ability to further their education and training. Argentine researchers surveyed demonstrated a strong interest and willingness to contribute their work and expertise to inform Argentine health policy development.

Conclusion

Despite Argentina’s long scientific tradition, there are relatively few institutionalized linkages between health research results and health policymaking. Based on the results of this study, the disconnect between political decision-making and the health research system, coupled with fewer opportunities for formalized or informal researcher/decision-maker interaction, contribute to the challenges in evidence informing health policymaking in Argentina. Improving personal contact and the building of relationships between researchers and policymakers in Argentina will require taking into account researcher perceptions of policymakers, as highlighted in this study.     

Systematic analysis of HD-ZIP transcription factors in sesame genome and gene expression profiling of SiHD-ZIP class I entailing drought stress responses at early seedling stage

Molecular Biology Reports - Sesame is an ancient oilseed crop, known for its high oil content and quality. Its sensitivity to drought at early seedling stage is one of the limiting factors...     

The mitochondrial protein C1qbp promotes cell proliferation,migration and resistance to cell death

Complement 1q-binding protein (C1qbp) is a mitochondrial protein reported to be upregulated in cancer. However, whether C1qbp plays a tumor suppressive or tumorigenic role in the progression of cancer is controversial. Moreover, the exact effects of C1qbp on cell proliferation, migration and death/survival have not been definitely proven. To this end, we comprehensively examined the effects of C1qbp on mitochondrial-dependent cell death, proliferation and migration in both normal and breast cancer cells using genetic gain- and loss-of-function approaches. In normal fibroblasts, overexpression of C1qbp protected the cells against staurosporine-induce apoptosis, increased proliferation, decreased cellular ATP and increased cell migration in a wound-healing assay. In contrast, the opposite effects were observed in fibroblasts depleted of C1qbp by RNA interference. C1qbp expression was found to be markedly elevated in 4 different human breast cancer cell lines as well as in ductal and adenocarcinoma tumors from breast cancer patients. Stable knockdown of C1qbp by shRNA in the aggressive MDA-MB-231 breast cancer cell line greatly reduced cell proliferation, increased ATP levels and decreased cell migration compared with control shRNA-transfected cells. Moreover, C1qbp knockdown elicited a significant increase in doxorubicin-induced apoptosis in the MDA-MB-231 cells. Finally, C1qbp upregulation was not restricted to breast cancer cells and tumors, as levels of C1qbp were also found to be significantly elevated in both human lung and colon cancer cell lines and carcinomas. Together, these results establish a pro-tumor, rather than antitumor, role for C1qbp and indicate that C1qbp could serve as a molecular target for cancer therapeutics.Key words: mitochondria, cell proliferation, cell migration, cell death, tumor cells