Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.     

Biosensors based on peptide exposure show single molecule conformations in live cells

1. Download : Download high-res image (175KB)
2. Download : Download full-size image

     

Tuning cellular responses to BMP-2 with material surfaces

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications.     

Fluid shear stress regulates HepG2 cell migration though time-dependent integrin signaling cascade

Hepatocellular carcinoma (HCC) is a subtype of malignant liver cancer with poor prognosis and limited treatment options. It is noteworthy that mechanical forces in tumor microenvironment play a pivotal role in mediating the behaviors and functions of tumor cells. As an instrumental type of mechanical forces in vivo, fluid shear stress (FSS) has been reported having potent physiologic and pathologic effects on cancer progression. However, the time-dependent mechanochemical transduction in HCC induced by FSS remains unclear. In this study, hepatocellular carcinoma HepG2 cells were exposed to 1.4 dyn/cm² FSS for transient duration (15s and 30s), short duration (5 min, 15 min and 30 min) and long duration (1h, 2h and 4h), respectively. The expression and translocation of Integrins induced FAK-Rho GTPases signaling events were examined. Our results showed that FSS endowed HepG2 cells with higher migration ability via reorganizing cellular F-actin and disrupting intercellular tight junctions. We further demonstrated that FSS regulated the expression and translocation of Integrins and their downstream signaling cascade in time-dependent patterns. The FSS downregulated focal adhesion components (Paxillin, Vinculin and Talin) while upregulated the expression of Rho GTPases (Cdc42, Rac1 and RhoA) in long durations. These results indicated that FSS enhanced tumor cell migration through Integrins-FAK-Rho GTPases signaling pathway in time-dependent manners. Our in vitro findings shed new light on the role of FSS acting in physiologic and pathological processes during tumor progression, which has emerged as a promising clinical strategy for liver carcinoma.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Fungal spore concentrations in the air at Tiruchirapalli (India) 1987–1988

Summary Using an aeroscope, airborne fungal spores were sampled for two years, 1987–1988 at Tiruchirapalli, Tamil Nadu, India. The aerospora components, their seasonal and annual variations in incidence in the air are discussed and a spore calendar for Tiruchirapalli is presented.     

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover

1. Download : Download high-res image (191KB)
2. Download : Download full-size image

     

Betulachrysoquinone hemiketal: A p-benzoquinone hemiketal macrocyclic compound produced by Phanerochaete chrysosporium

Betulachrysoquinone hemiketal was isolated from pre-extracted wood of Betula lutea Michx. inoculated with Phanerochaete chrysosporium Burds. Acid-catalysed hydrolysis of betulachrysoquinone hemiketal produced betulachrysoquinone which was shown to be 2-hydroxy-6-(13′-hydroxytetradecanyl)-p-benzoquinone.     

An improved method for light- and electron microscopial studies of nematode/fungal interactions

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.