Basic Fibroblast Growth Factor Receptor Research Articles

Unveiling the mechanism of Buddleja officinalis against esophageal squamous cell carcinoma through network pharmacology and molecular docking approaches

In this research, we aim to explore the underlying mechanism of Buddleja officinalis (BO) in inhibiting esophageal squamous cell carcinoma (ESCC) by means of network pharmacology and molecular docking approaches. First, BO component targets were determined from the Traditional Chinese Medicine Systematic Pharmacology and HERB databases (known as BenCaoZuJian in Chinese transliteration), and ESCC disease targets were identified from GeneCards and DisGeNET databases. Second, the Venny 2.1 online tool was utilized to visualize the intersection targets, and shared potential targets between BO and ESCC were identified using the STRING database. Third, the component-target-pathway networks were constructed using Cytoscape software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were utilized for further analyzing the mechanism of BO in inhibiting ESCC. Finally, molecular docking technique was employed to delineate the docking profiles of BO and determine the optimal active component, which is threonine protein kinase (AKT1). We screened six active components and 227 targets from BO, of which 24 were shared targets of ESCC and BO. The network pharmacology analysis indicated core targets with high degrees, namely, serum albumin, insulin-like growth factor 1 receptor, AKT1, estrogen receptor, and basic fibroblast growth factor receptor 1, which are the most likely binding sites for the active components in BO. The related signaling pathways underpinning the inhibition of ESCC by BO encompass MAPK signaling pathway, adhesion junction pathway, and gastric cancer pathway. Moreover, linarin was recognized as the most suitable component for AKT1. Our results revealed that BO exhibits multicomponent, multi-target, and multi-pathway characteristics, which offer a scientific foundation for elucidating its therapeutic mechanism in ESCC and present novel insights for future investigations.

Features of Intracellular Signal Transduction in Neural Stem Cells under the Influence of Alkaloid Songorine, an Agonist of Fibroblast Growth Factor Receptors.

We performed a comparative in vitro study of the involvement of NF-κB, PI3K, cAMP, ERK1/2, p38, JAKs, STAT3, JNK, and p53-dependent intracellular signaling in the functioning of neural stem cells (NSC) under the influence of basic fibroblast growth factor (FGF) and FGF receptor agonist, diterpene alkaloid songorine. The significant differences in FGFR-mediated intracellular signaling in NSC were revealed for these ligands. In both cases, stimulation of progenitor cell proliferation occurs with the participation of NF-κB, PI3K, ERK1/2, JAKs, and STAT3, while JNK and p53, on the contrary, inhibit cell cycle progression. However, under the influence of songorin, cAMP- and p38-mediated cascades are additionally involved in the transmission of the NSC division-activating signal. In addition, unlike FGF, the alkaloid stimulates progenitor cell differentiation by activating ERK1/2, p38, JNK, p53, and STAT3.

Benchmarking Water Models in Molecular Dynamics of Protein-Glycosaminoglycan Complexes.

Glycosaminoglycans (GAGs) made of repeating disaccharide units intricately engage with proteins, playing a crucial role in the spatial organization of the extracellular matrix (ECM) and the transduction of biological signals in cells to modulate a number of biochemical processes. Exploring protein-GAG interactions reveals several challenges for their analysis, namely, the highly charged and periodic nature of GAGs, their multipose binding, and the abundance of the interfacial water molecules in the protein-GAG complexes. Most of the studies on protein-GAG interactions are conducted using the TIP3P water model, and there are no data on the effect of various water models on the results obtained in molecular dynamics (MD) simulations of protein-GAG complexes. Hence, it is essential to perform a systematic analysis of different water models in MD simulations for these systems. In this work, we aim to evaluate the properties of the protein-GAG complexes in MD simulations using different explicit: TIP3P, SPC/E, TIP4P, TIP4PEw, OPC, and TIP5P and implicit: IGB = 1, 2, 5, 7, and 8 water models to find out which of them are best suited to study the dynamics of protein-GAG complexes. The FF14SB and GLYCAM06 force fields were used for the proteins and GAGs, respectively. The interactions of several GAG types, such as heparin, chondroitin sulfate, and hyaluronic acid with basic fibroblast growth factor, cathepsin K, and CD44 receptor, respectively, are investigated. The observed variations in different descriptors used to study the binding in these complexes emphasize the relevance of the choice of water models for the MD simulation of these complexes.

Gentiopicroside injection promotes the healing of pressure injury wounds by upregulating the expression of bFGFR1.

To observe the therapeutic effect of gentiopicroside, as the main component of Gentianaceae, on wounds in pressure injury (PI) model rats and explore its mechanism. Male Sprague Dawley rats were randomly divided into control group, model group and gentiopicroside groups (50, 100 and 200 mg·kg-1·d-1 for 9 consecutive days). The mice's skeletal muscle fibroblast line NOR-10 cells were collected after being treated with gentiopicroside (0.2~5.0 M) and basic fibroblast growth factor receptor 1 (bFGFR1) inhibitor (5.0 M SU5402) for 7 days. Compared to the model group, the gentiopicroside groups showed significantly increased wound healing rates, reduced inflammatory cells in the wound tissues, and significantly increased expression levels of proliferating cell nuclear antigen (PCNA) and bFGFR1, accompanied by increased proliferation of new myofibroblasts. Gentiopicroside upregulated the mRNA expression of bFGFR1 and PCNA in NOR-10 cells in a dose-dependent manner; however, SU5402 reversed the effect of gentiopicroside. Gentiopicroside may promote myofibroblast proliferation by upregulating the expression of bFGFR1 and PCNA and ultimately accelerating the healing of PI wounds.

Study of aqueous humour inflammatory mediators’ levels in a cohort of Egyptian patients with diabetic macular oedema

BackgroundThe aim was to study aqueous humour inflammatory mediators’ levels in a cohort of Egyptian patients with diabetic macular oedema (DMO).MethodsThis was a case-control prospective study conducted on 2 groups: 25 eyes of 22 (11 females) patients seeking treatment for DMO as patients group, and 10 eyes of 10 (4 females) cataract patients as a control group. Aqueous humour was aspirated before intravitreal injection (patients’ group) or cataract surgery (control group). Inflammatory mediators in aqueous humour were measured using a multiplex bead immunoassay kit of 27 pre-mixed cytokines.ResultsEotaxin, interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleukin-8 (IL-8/CXCL8) were found significantly higher in patients’ group compared to control group (p = 0.043, 0.037, 0.001, 0.015 respectively). On the contrary, interferon-gamma (IFN-gamma) and granulocyte colony-stimulating factor (G-CSF) were found significantly higher in control group than patients’ group (p = 0.003, 0.019 respectively). Basic fibroblast growth factor (Basic-FGF/FGF-2) and interleukin-1 receptor antagonist (IL-1ra) were found higher (but not statistically significant) in controls (p = 0.100 and 0.070 respectively). Additionally, a negative and significant correlation was found between Eotaxin level in aqueous humour and central macular thickness.ConclusionsSome mediators might be implicated in the pathogenesis of DMO either augmenting or suppressing role. Eotaxin, IP-10, MCP-1 and IL-8 might have a role in cases not responding to standard anti-vascular endothelial growth factor (VEGF) therapy. IL-1ra might have a protective role; therefore, the effectiveness of intravitreal injection of IL-1ra homologue needs to be studied in future clinical trials.

Sapropterin reduces coronary artery malformation in offspring of pregestational diabetes mice

Endothelial nitric oxide synthase (eNOS) and oxidative stress are critical to embryonic coronary artery development. Maternal diabetes increases oxidative stress and reduces eNOS activity in the fetal heart. Sapropterin (Kuvan®) is an orally active, synthetic form of tetrahydrobiopterin (BH4) and a co-factor for eNOS with antioxidant properties. The aim of the present study was to examine the effects of sapropterin on fetal coronary artery development during pregestational diabetes in mice. Diabetes was induced by streptozotocin to adult female C57BL/6 mice. Sapropterin (10 mg/kg/day) was orally administered to pregnant mice from E0.5 to E18.5. Fetal hearts were collected at E18.5 for coronary artery morphological analysis. Sapropterin treatment to diabetic dams reduced the incidence of coronary artery malformation in offspring from 50.0% to 20.6%. Decreases in coronary artery luminal diameter, volume and abundance in fetal hearts from diabetic mothers, were prevented by sapropterin treatment. Maternal diabetes reduced epicardial epithelial-to-mesenchymal transition (EMT) and expression of transcription and growth factors critical to coronary artery development including hypoxia-inducible factor 1a (Hif1a), Snail1, Slug, β-catenin, retinaldehyde dehydrogenase 2 (Aldh1a2), basic fibroblast growth factor (bFGF) and vascular endothelial group factor receptor 2 (Vegfr2) in E12.5 hearts. Additionally, eNOS phosphorylation was lower while oxidative stress was higher in E12.5 hearts from maternal diabetes. Notably, these abnormalities were all restored to normal levels after sapropterin treatment. In conclusion, sapropterin treatment increases eNOS activity, lowers oxidative stress and reduces coronary artery malformation in offspring of pregestational diabetes. Sapropterin may have therapeutic potential in preventing coronary artery malformation in maternal diabetes.

P4493Microvesicles from patients with pulmonary arterial hypertension modulate bFGF from endothelial cells

Abstract Background Pulmonary Arterial Hypertension (PAH) is a progressive condition with remodeling of precapillary arteries. Cytokines involved in angiogenesis have been found elevated in PAH but the role of proangiogenic factors and their regulation is far from fully understood. Microvesicles (MVs) can act as intercellular messengers and modify cell production of proteins. Purpose To evaluate whether MVs from patients with PAH modulate proangiogenic factors in plasma and endothelial cells. Methods 62 patients with PAH and 20 healthy controls were enrolled in the study. The number of MVs were determined in whole blood by flow cytometry. For in vitro studies, MVs were isolated from plasma and a primary endothelial cell line of human pulmonary artery endothelial cells between passages 3 to 7 were used. Protein levels in plasma and conditioned media were analyzed by electrochemiluminescence whereas protein levels in cell lysates were analyzed by western blot (WB). Results The majority of the MVs in whole blood were derived from platelets and patients with PAH had higher concentrations compared to healthy controls (p<0.0001). In plasma both basic Fibroblast Growth Factor (bFGF) and vascular endothelial growth factor receptor 1 (Flt-1) were increased in the patients as compared to healthy controls (median in pg/ml (IQR) Pat. vs Ctrl: bFGF 4.2 (2.5–9.2) vs 1.2 (0.9–2.1); Flt-1 143.0 (88.5–234.5) vs 53.9 (47.7–59.67); p<0.001 for both). Endothelial cells were incubated with or without MVs for 18 hours and effects were monitored by analyzing eNOS and ICAM-1 levels by WB. Both markers were significantly altered, i.e. an about 50% decrease of eNOS and a 50% increase of ICAM-1, by patient MVs but not by the control MVs. The endothelial cells were then incubated with MVs for 6h, 12h, 24h, and 48h and bFGF and Flt-1 were measured in the conditioned media. The presence in media of both proteins were increased over time, but only bFGF was significantly regulated by MVs from PAH patient as compared to MVs from the control group at 24h (2.8 fold increase) and at 48h (12.7 fold increase). Conclusions An increase of two important proangiogenic proteins were found in PAH patients. The MVs from these patients modulate the endothelial response and contribute to a release of bFGF, whereas the Flt-1 levels are regulated by other currently unknown mechanisms. Further studies of the MVs from PAH patients are needed to elucidate how they are involved in the angiogenic process. Acknowledgement/Funding Swedish Research Council, Science For Life Laboratory Sweden

Construction of a human monoclonal antibody against bFGF for suppression of NSCLC.

Compelling evidence implicates that overexpression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) drives tumor progression, can serve as prognostic biomarkers or therapeutic targets for NSCLC patients. But at present, we still lack of effective drugs for bFGF. The preparation of monoclonal antibodies against bFGF or to understand its mechanism of action is urgently need. Previously, we used hybridoma technology to produce a murine anti-bFGF monoclonal antibody (E12). However, E12 carries risks of heterogeneity and immunogenicity. In the present work, we produced three humanized variants (H1L1, H2L2 and H3L3) based on E12 by substituting residues in or near the complementarity-determining region (CDR). In addition, we thoroughly explored VH/VL domain combinations to simulate full-length IgG1 antibodies using computational protein design. H3L3 was selected for further study, as it demonstrated the best humanization and strongest affinity for bFGF. Specially, humanization of H3L3's light chain and heavy chain were 100% and 98.89%, respectively. The FGF2 neutralizing effect of H3L3 were confirmed by ELISA. We also found that H3L3 can effectively suppress the growth and angiogenesis of cancer through reduce the phosphorylation of AKT and MAPK. Moreover, H3L3 dramatically reduced tumor size and micro-vessel density in nude mice. Altogether, our study demonstrates that H3L3 exerts anti-tumor effects by impeding NSCLC development.

Industrial hog farming is associated with altered circulating immunological markers

ObjectivesThe previously observed inverse association between hog farming and risk of lung cancer in the Agricultural Health Study (AHS) has been attributed to endotoxin exposure, the levels of which are...

Production of bFGF monoclonal antibody and its inhibition of metastasis in Lewis lung carcinoma.

Basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor 1 (FGFR1) are associated with drug resistance in lung cancer. In the present study, mouse monoclonal antibodies (mAb) against human bFGF, targeting the binding site of bFGF with FGFR1 were produced, and the antitumor activity and inhibition of metastasis was studied in Lewis lung carcinoma (LLC). A total of four hybridoma cell strains that stably secreted bFGF mAb were obtained. mAbE12 was selected as the most effective for use in the following studies, with a relative affinity constant of 5.66×108 l/mol. mAbE12 was demonstrated to inhibit cell proliferation and tumor growth in vitro and in vivo. Furthermore, mAbE12 blocked migration and metastasis of LLC cells in vitro and in vivo. This occurred due to a mAbE12-induced upregulation of E-cadherin expression through the protein kinase B-glycogen synthase kinase 3 β-Snail pathway. These results suggested that mAbE12 may be a potential antibody for the treatment of lung cancer.

Optimal FSH usage in revascularization of allotransplanted ovarian tissue in mice

BackgroudOvarian transplantation is a useful method for preserving the fertility of young women with cancer who undergo radiotherapy and chemotherapy. Follicle-stimulating hormone (FSH) is use to protect transplanted ovarian tissues from ischemia injury through promoting revascularization after transplantation, but the side effect of high level FSH is ovarian overstimulation leading to substantial follicular loss. In this study, we investigated the optimal usage of FSH on revascularization in the in vitro cultured ovarian tissues before and after transplantation.ResultsFSH mainly exhibited an additive response in the gene and protein expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and follicle stimulating hormone receptor (FSHR) with its raised concentrations (0.15 IU/ml, 0.30 IU/ml and 0.60 IU/ml) and prolonged treatment (3 h, 6 h, 12 h, 24 h). The concentrations with 0.60 IU/ml FSH could obviously promoted the expression of VEGF, bFGF and FSHR, but under this concentration FSH could also overstimulated the ovarian tissue leading to follicular loss. With the increase of culture time, the gene and protein expression of VEGF and bFGF both were up-regulated in all of the FSH added groups, but FSHR expression decreased when culture time exceeded 12 h. So we chose 0.30 IU/ml FSH added concentration and 6 h culture time as the FSH usage condition in functional revascularization verification experiment, and found that under this condition FSH promoted 2.5 times increase of vascular density in treated group than in control group after ovarian tissues transplantation.ConclusionOvarian intervention with 0.30 IU/ml FSH for 6 h is an optimal FSH usage condition which could accelerate the revascularization in the allotransplanted ovarian tissue and can not produce ovarian overstimulation.

Stephania Tetrandra and Ginseng-Containing Chinese Herbal Formulation NSENL Reverses Cisplatin Resistance in Lung Cancer Xenografts.

Chinese Herbal Formulation, supplement energy and nourish lung (SENL), effectively enhances chemotherapeutic efficacy in lung cancer treatment and reverses multi-drug resistance (MDR) in lung cancer cells in vitro. The present study is designed to assess the effect of a New SENL (NSENL, modification of SENL) formulation on resistance to chemotherapy of cisplatin (DDP)-resistant human lung cancer cell line (A549/DDP) xenografts in nude mice. We assessed six constituents in NSENL by high performance liquid chromatography (HPLC). BALB/c nude mice harboring A549/DDP cell xenografts were established to assess the antitumor effect of NSENL and its impact on the expression of MDR related genes. The six constituents in NSENL, including ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rg3, astragaloside IV, ophiopogonin D and tetrandrine were quantitated simultaneously by HPLC. The combination of NSENL with DDP significantly inhibited tumor growth at a rate of up to 66.8% ([Formula: see text]). In addition, NSENL as monotherapy or combined with DDP downregulated multidrug resistance-associated protein 1 (MRP1), basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor 1 (FGFR1) at both the mRNA and protein levels ([Formula: see text]), reduced glutathione S-transferase π (GST-π) protein expression and tumor microvascular density as well as decreased phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) ([Formula: see text]). These findings demonstrated that NSENL can reverse MDR in A549/DDP cells in vivo, an effect possibly associated with downregulation of MDR-associated genes as well as inhibition of bFGF/FGFR and phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathways.

Inorganic polyphosphate triggers upregulation of interleukin 11 in human osteoblast-like SaOS-2 cells

Polyphosphate (polyP) is abundant in bone but its roles in signaling and control of gene expression remain unclear. Here, we investigate the effect of extracellular polyP on proliferation, migration, apoptosis, gene and protein expression in human osteoblast-like SaOS-2 cells. Extracellular polyP promoted SaOS-2 cell proliferation, increased rates of migration, inhibited apoptosis and stimulated the rapid phosphorylation of extracellular-signal-regulated kinase (ERK) directly through basic fibroblast growth factor receptor (bFGFR). cDNA microarray revealed that polyP induced significant upregulation of interleukin 11 (IL-11) at both RNA and protein levels.

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Lithium's gene expression profile, relevance to neuroprotection A cDNA microarray study.

Lithium can prevent 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) dopaminergic neurotoxicity in mice. This is attributed to induced antioxidant and antiapoptotic state, which among other factors results from induction of Bcl-2 and reduction of Bax, however, cDNA microarray reveals that this represents only one cascade of lithium targets. From analyzing the gene expression profile of lithium, we are able to point out candidate genes that might be involved in the antioxidant and neuroprotective properties of lithium. Among these are, the cAMP response element binding (CREB) protein, extracellular signal-regulated kinase (ERK), both CREB and ERK-part of the mitogen-activated kinase pathway-were upregulated by lithium, downregulated by MPTP, and maintained in mice fed with lithium chloride (LiCl) supplemented diet and treated with MPTP. Our positive control included tyrosine hydroxylase which both its mRNA and protein levels were independently measured, in addition to Bcl-2 protein levels. Other important genes which were similarly regulated are plasma glutathione peroxidase precursor (GSHPX-P), protein kinase C alpha type, insulin-like growth factor binding protein 4 precursor, and interferon regulatory factor. In addition, some genes were oppositely regulated, i.e., downregulated by lithium, upregulated by MPTP, and maintained in mice fed with LiCl supplemented diet and treated with MPTP, among these genes were basic fibroblast growth factor receptor 1 precursor, inhibin alpha subunit, glutamate receptor subunit zeta 1 precursor (NMD-R1), postsynaptic density protein-95 which together with NMD-R1 can form an apoptotic promoting complex. The discussed targets represent part of genes altered by chronic lithium. In fact lithium affected the expressions of more than 50 genes among these were basic transcription factors, transcription activators, cell signaling proteins, cell adhesion proteins, oncogenes and tumor suppressors, intracellular transducers, survival and death genes, and cyclins, here we discuss the relevance of these changes to lithium's reported neuroprotective properties.

S100B Engages RAGE or bFGF/FGFR1 in Myoblasts Depending on Its Own Concentration and Myoblast Density. Implications for Muscle Regeneration

In high-density myoblast cultures S100B enhances basic fibroblast growth factor (bFGF) receptor 1 (FGFR1) signaling via binding to bFGF and blocks its canonical receptor, receptor for advanced glycation end-products (RAGE), thereby stimulating proliferation and inhibiting differentiation. Here we show that upon skeletal muscle injury S100B is released from myofibers with maximum release at day 1 post-injury in coincidence with satellite cell activation and the beginning of the myoblast proliferation phase, and declining release thereafter in coincidence with reduced myoblast proliferation and enhanced differentiation. By contrast, levels of released bFGF are remarkably low at day 1 post-injury, peak around day 5 and decline thereafter. We also show that in low-density myoblast cultures S100B binds RAGE, but not bFGF/FGFR1 thereby simultaneously stimulating proliferation via ERK1/2 and activating the myogenic program via p38 MAPK. Clearance of S100B after a 24-h treatment of low-density myoblasts results in enhanced myotube formation compared with controls as a result of increased cell numbers and activated myogenic program, whereas chronic treatment with S100B results in stimulation of proliferation and inhibition of differentiation due to a switch of the initial low-density culture to a high-density culture. However, at relatively high doses, S100B stimulates the mitogenic bFGF/FGFR1 signaling in low-density myoblasts, provided bFGF is present. We propose that S100B is a danger signal released from injured muscles that participates in skeletal muscle regeneration by activating the promyogenic RAGE or the mitogenic bFGF/FGFR1 depending on its own concentration, the absence or presence of bFGF, and myoblast density.

The Evaluation of Basic Fibroblast Growth Factor and Fibroblastic Growth Factor Receptor 1 Levels in Saliva and Serum of Patients with Salivary Gland Tumor

Basic fibroblast growth factor (FGF2) is a well-known endothelial mitogen that regulates endothelial cell proliferation, migration, differentiation, and survival. In the present study, we investigated the levels of FGF2 and fibroblastic growth factor receptor 1 (FGFR1) in saliva and serum of patients with salivary gland tumors. Saliva and serum samples were collected from 43 patients with salivary gland tumors and 40 healthy volunteers. The FGF2 and FGFR1 concentrations in saliva and serum samples were measured by enzyme-linked immunosorbent assay. We found that the levels of FGF2 and FGFR1 in saliva and serum from patients with salivary gland tumors were significantly higher than those from healthy control subjects. These results suggest that salivary FGF2 and FGFR1 can be used as potential biomarkers in the diagnosis of salivary gland tumors.

Another Step Toward the Cure of Metastatic Renal Cell Carcinoma?

Another Step Toward the Cure of Metastatic Renal Cell Carcinoma?

Long-Term Culture and Transplantation of Spermatogonial Stem Cells from BALB/c Mice

Development of a culture system that supports self-renewal and proliferation of spermatogonial stem cells (SSCs) is enormously valuable for experimental research and potential treatment for male infertility. Although several research groups had reported their successes in SSC isolation and culture, the two current accepted culture systems are different in cell enrichment methods, serum and growth factors. Previous researches also indicated SSCs from different mouse strains required different culture conditions. Here we report for the first time that SSCs from BALB/c mice could be cultured in an improved culture system for 3 months. The modified culture system consisted of an improved enzymatic procedure, the enrichment of undifferentiated spermatogonia by differential adherence selection of isolated SSCs, mouse embryonic fibroblast feeder cells, StemPro-34 SFM medium supplemented with glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor and GDNF-family receptor α1 (GFRα1). The improved digestion method increased the viability and enrichment efficiency of isolated testis cells. Furthermore, basal culture medium with 10% fetal bovine serum as selected medium could increase the number of germ cell colonies in the initiation stage of culture. Cultured SSCs were characterized morphologically and formed typical colonies. Immunocytochemical staining and RT-PCR showed that cultured SSCs expressed Oct-4, GFRα1, Sox2 and several other special genes resembling undifferentiated spermatogonia. Spermatogonia transplantation further confirmed that cultured SSCs were functionally normal and could restore complete spermatogenesis. The culture methods described here could serve as a paradigm to establish conditions for the culture of SSCs from other species, allowing identification of universal factors necessary for proliferation of SSCs.

The many faces of S100B protein: when an extracellular factor inactivates its own receptor and activates another one

The Ca(2+)-binding protein of the EF-hand type, S100B, is an intracellular regulator and an extracellular signal. Within cells S100B interacts with several proteins thereby regulating energy metabolism, Ca2+ homeostasis, protein phosphorylation and degradation, and cell locomotion, proliferation and differentiation. Once secreted/released, S100B exerts autocrine and paracrine effects on responsive cells by engaging the receptor for advanced glycation end products. However, recent evidence suggests that S100B might also activate basic fibroblast growth factor receptor 1 via prior binding to basic fibroblast growth factor.