In regard to erectile function, Yin is flaccidity and Yang erection. In the past decade, research has mostly focused on the Yang aspect of erectile function. However, in recent years, the Yin side is attracting increasingly greater attention. This is due to the realization that penile flaccidity is no less important than penile erection and is actively maintained by mechanisms that play critical roles in certain types of erectile dysfunction (ED); for example, in diabetic patients. In addition, there is evidence that the Yin and Yang signaling pathways interact with each other during the transition from flaccidity to erection, and vice versa. As such, it is important that we view erectile function from not only the Yang but also the Yin side. The purpose of this article is to review recent advances in the understanding of the molecular mechanisms that regulate the Yin and Yang of the penis. Emphasis is given to the Rho kinase signaling pathway that regulates the Yin, and to the cyclic nucleotide signaling pathway that regulates the Yang. Discussion is organized in such a way so as to follow the signaling cascade, that is, beginning with the extracellular signaling molecules (e.g., norepinephrin and nitric oxide) and their receptors, converging onto the intracellular effectors (e.g., Rho kinase and protein kinase G), branching into secondary effectors, and finishing with contractile molecules and phosphodiesterases. Interactions between the Yin and Yang signaling pathways are discussed as well.
Erectile Dysfunction ; Humans ; Male ; Penile Erection

The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.
Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; Cell Survival ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Fetus ; Flow Cytometry ; Homeodomain Proteins ; analysis ; Humans ; Hyaluronan Receptors ; analysis ; Immunohistochemistry ; Integrin beta1 ; analysis ; Islets of Langerhans ; Proliferating Cell Nuclear Antigen ; analysis ; Stem Cells ; cytology ; metabolism ; Trans-Activators ; analysis

Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra 8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse, Stra 8 is restrictively expressed in male germ line cells, and its function is related to the development of sperm. In order to investigate the feature of Stra 8 gene expression,the 1.4 kb (-1407 - +7) promoter region of Stra 8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4 kb promoter region, and the reporter gene of EGFP that was regulated by 1.4kb Stra 8 promoter. To investigate the specificity of Stra 8 promoter,the vector pStro-EGFP was transfected into undifferentiated mouse stem cells such as ES-129, bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results showed that the expression of GFP was only observed in the mSSC cells,which indicated that Stra 8 gene was specially regulated in testis tissue. As the gene marker,vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After being selected by G418 for 2 weeks,the mMSC cells were induced by retinoic acid. After 12 hours induction, some induced cells started to express GFP protein, which was observed under the fluorescence microscope. At the same time, several stem cell specificity biomarkers such as Oct4, and spermatogonial stem cell biomarkers such as CyclinA2 and Stra 8 were detected in the induced cells by RT-PCR method. These results showed that the mMSCs would differentiated to spermatognial stem cells after induced by Retinoic Acid.
Adaptor Proteins, Signal Transducing ; Animals ; Cell Differentiation ; genetics ; Cells, Cultured ; Gene Expression ; Male ; Mice ; Promoter Regions, Genetic ; Proteins ; genetics ; Spermatogonia ; cytology ; metabolism ; Stem Cells ; cytology ; metabolism ; Tretinoin ; pharmacology

We developed phase-switch microfluidic devices for molecular profiling of a large number of single cells. Whole genome microarrays and RNA-sequencing are commonly used to determine the expression levels of genes in cell lysates (a physical mix of millions of cells) for inferring gene functions. However, cellular heterogeneity becomes an inherent noise in the measurement of gene expression. The unique molecular characteristics of individual cells, as well as the temporal and quantitative information of gene expression in cells, are lost when averaged among all cells in cell lysates. Our single-cell technology overcomes this limitation and enables us to obtain a large number of single-cell transcriptomes from a population of cells. A collection of single-cell molecular profiles allows us to study carcinogenesis from an evolutionary perspective by treating cancer as a diverse population of cells with abnormal molecular characteristics. Because a cancer cell population contains cells at various stages of development toward drug resistance, clustering similar single-cell molecular profiles could reveal how drug-resistant sub-clones evolve during cancer treatment. Here, we discuss how single-cell transcriptome analysis technology could enable the study of carcinogenesis from an evolutionary perspective and the development of drug-resistance in leukemia. The single-cell transcriptome analysis reported here could have a direct and significant impact on current cancer treatments and future personalized cancer therapies.
Carcinogenesis ; genetics ; Drug Resistance, Neoplasm ; Gene Expression Profiling ; Hematopoietic Stem Cells ; metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; pathology ; Microfluidic Analytical Techniques ; Single-Cell Analysis ; methods ; Transcriptome

BACKGROUNDCardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease. Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum (ER) stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) for five days as an in vitro model, to investigate if acetylated low-density lipoprotein (AcLDL) loading could also induce apoptosis and its underlying mechanisms.

METHODSOil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol (FC) and cholesterol ester (CE) assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed.

RESULTSConfocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 microg/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER.

CONCLUSIONER stress is involved in AcLDL induced apoptosis in THP-1 macrophages with free cholesterol accumulation in the ER.

Apoptosis ; drug effects ; Blotting, Western ; Cell Differentiation ; physiology ; Cell Line ; Cholesterol ; metabolism ; Chromatography, High Pressure Liquid ; Endoplasmic Reticulum ; metabolism ; Humans ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; drug effects ; Microscopy, Confocal ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Transcription Factor CHOP ; genetics

OBJECTIVE This study aimed to determine the relationship between individual allergens with current wheezing and bronchial hyperresponsiveness (BHR) in schoolchildren from three chinese cities: Beijing, Guangzhou and Hong Kong. METHODS Community-based random samples of 10-yr-old schoolchildren from the 3 cities were recruited for study using the International Study of Asthma and Allergies in Childhood (ISAAC) Phase II protocol. The subjects were studied by parental questionnaires (n = 10,902), skin-prick tests (n = 3478), and methacholine challenge tests (n = 608). RESULTS The highest prevalence rates of wheezing in the past 12 months (Beijing, 3.8%; Guangzhou, 3.4%; Hong Kong, 5.8%) and atopy (Beijing, 23.9%; Guangzhou, 30.8%; Hong Kong, 41.2%, defined as having Allergens ; classification ; immunology ; Asthma ; etiology ; immunology ; Bronchial Hyperreactivity ; etiology ; immunology ; Child ; China ; Hong Kong ; Humans ; Risk Factors ; Skin Tests ; Surveys and Questionnaires

BACKGROUNDLow-density lipoprotein (LDL) receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line (HepG2) cells under physiological and inflammatory conditions.

METHODSIntracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum (ER) to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies.

RESULTSLDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1 beta (IL-1 beta) further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1 beta caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium.

CONCLUSIONSIL-1 beta disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.

Cell Line, Tumor ; Cholesterol ; analysis ; Endoplasmic Reticulum ; metabolism ; Feedback, Physiological ; Humans ; Interleukin-1beta ; pharmacology ; Intracellular Signaling Peptides and Proteins ; analysis ; genetics ; Membrane Proteins ; analysis ; genetics ; Protein Transport ; RNA, Messenger ; analysis ; Receptors, LDL ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sterol Regulatory Element Binding Protein 2 ; analysis ; genetics

AIMTo identify proteins that are differentially expressed in cells derived from normal and diseased tunica albuginea (TA) as related to Peyronie's disease (PD).

METHODSCells with characteristics of fibroblasts were isolated from two tissue sources. Those from the plaque of patients with PD were designated as PT cells, and those from the normally-appearing TA of the same patients were designated as NT cells. Messenger RNAs of these cells were analyzed by real-time polymerase chain reaction (RT-PCR) for the expression of monocyte chemoattractant protein 1 (MCP-1). Crude protein lysates were analyzed by surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) with IMAC30-Cu, CM10, and H50 chips. Each lysate was then separated into six fractions, which were further analyzed by SELDI-MS.

RESULTSRT- PCR analysis showed that PT cells expressed higher levels of MCP-1 than their counterpart NT cells. SELDI-MS analysis showed that the crude protein lysates of all four cell strains produced similar and reproducible protein profiles on IMAC30-Cu and CM10 chips. Additional SELDI-MS analyses with the fractionated lysates detected three proteins of 11.6 kDa, 14.5 kDa, 22.6 kDa that were upregulated in PT cells and two proteins of 6.3 kDa and 46.9 kDa that were downregulated in PT cells.

CONCLUSIONMCP-1, which is often involved in tissue fibrosis, was expressed at higher levels in PT than that in NT cells. Five potential biomarkers for PD were identified by SELDI-MS analysis.

Base Sequence ; Biomarkers ; analysis ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; DNA Primers ; Humans ; Male ; Mass Spectrometry ; methods ; Penile Induration ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Male germ stem cells (mGSCs), which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells, and the cells were directional induced to formaactin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells.
Animals ; Cattle ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Fetus ; cytology ; Male ; Pluripotent Stem Cells ; cytology ; Spermatozoa ; cytology

Stem cells hold great promise for regenerative medicine because of their ability to self-renew and to differentiate into various cell types. Although embryonic stem cells (BSC) have greater differentiation potential than adult stem cells, the former is lagging in reaching clinical applications because of ethical concerns and governmental restrictions. Bone marrow stem cells (BMSC) are the best-studied adult stem cells (ASC) and have the potential to treat a wide variety of diseases, including erectile dysfunction (ED) and male infertility. More recently discovered adipose tissue-derived stem cells (ADSC) are virtually identical to bone marrow stem cells in differentiation and therapeutic potential, but are easier and safer to obtain, can be harvested in larger quantities, and have the associated benefit of reducing obesity. Therefore, ADSC appear to be a better choice for future clinical applications. We have previously shown that ESC could restore the erectile function of neurogenic ED in rats, and we now have evidence that ADSC could do so as well. We are also investigating whether ADSC can differentiate into Leydig, Sertoli and male germ cells. The eventual goal is to use ADSC to treat male infertility and testosterone deficiency.
Adult Stem Cells ; Animals ; Cell Transplantation ; Embryonic Stem Cells ; Erectile Dysfunction ; therapy ; Humans ; Infertility, Male ; therapy ; Male ; Research