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.     

Examination of Induced Sputum In the Diagnosis of Pneumocystis Carinii Pneumonia

The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Presence of serum albumin in normal human epidermis: Possible implications for the nutrition and physiology of stratified epithelia

In this paper, using both immunofluorescence and protein biochemistry techniques, we present definitive evidence that plasma proteins such as albumin are present within normal human epidermis. This result confirms several previous reports supporting the idea that relatively large molecules can diffuse through the epidermal basement membrane into epidermis. Our results bring new insights for discussing how hydrophobic ligands or drugs present in the bloodstream and bound to plasmatic carriers can reach epidermal cells of all layers.Abbreviations CHAPS 3-[3-cholamidopropyl dimethylammonio] propane sulfonate - kD kilodaltons - BSA bovine serum albumin - 2ME 2-mercaptoethanol - DTT dithiothreitol - SDS sodium dodecyl sulfate - pI isoelectric point - Mw molecular weight - Tris Tris-(hydroxymethyl) aminomethane - 1D one dimensional - 2D two dimensional - PAGE poly acrylamide gel electrophoresis - MEM Minimal Eagle's Medium     

Levels and Distribution of the Calcium-Modulated Proteins S100 and Calmodulin in Rat C6 Glioma Cells

To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium-modulated proteins, we have examined the calcium-modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100 beta polypeptide. The level of S100 beta in C6 cells increases approximately 20-fold from subconfluency to postconfluency whereas the level of calmodulin increases only about two-fold. The subcellular distribution of S100 beta and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100- and calmodulin-binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100 beta is the predominant S100 polypeptide in C6 cells and suggest that changes in S100 beta and S100 beta-binding proteins may be involved in regulating S100-mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells.     

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: functional heterogeneity in cell subpopulations recognized by a monoclonal antibody

A hemocyte surface membrane marker (BGH₁) has been identified using hemocyte-specific monoclonal antibodies (mABs) generated by somatic cell fusion methods. The BGH₁ epitope was expressed on a subpopulation of circulating, glass-adherent blood cells from two strains of the snail, Biomphalaria glabrata. Approximately 40% of the circulating hemocytes from the PR albino (M-line) B. glabrata strain were BGH₁^?, compared to a prevalence of 10% BGH₁⁺ cells in the 10-R2 snail strain. When hemocytes were firmly attached and spread on a glass surface, BGH₁⁺ cells were morphologically distinguishable from BGH₁^? cells by their ovoid shape and the presence of short, thin filopodial projections along the ectoplasmic border. In contrast, BGH₁^? hemocytes were more pleomorphic and possessed long, spike-like filopodia. Moreover, the BGH₁ epitope was trypsin-resistant and retained its antigenic reactivity with probe mABs following fixation with paraformaldehyde or paraformaldehyde/MeOH. Fixation with glutaraldehyde, however, significantly reduced mAB binding to the BGH₁ surface epitope. There was no apparent age-dependent expression of the BGH₁ determinant since circulating hemocyte populations in very young (1–2 mm) to adult (10–12 mm) snails were composed of both BGH₁⁺ and BGH₁^? subpopulations. Quantitative shifts in the prevalence of epitope-bearing hemocytes between the smallest snail size class (1–2 mm) and the larger snails (3–4 and 10–12 mm) are believed to be due to a differential production and/or release of BGH₁^? hemocytes within the blood circulation rather than a gradual age-related change in the expression of surface antigens on individual cells. Experiments designed to assess the in vitro phagocytic capability and lysosomal acid phosphatase (APase) activity of mAB-reactive hemocytes revealed that BGH₁⁺ cells, when compared to those lacking the surface marker, were significantly reduced in both their phagocytic and APase-producing activities. Since the PR albino strain of B. glabrata possesses a higher proportion of BGH₁^? hemocytes and a lower total concentration of circulating cells than do snails of the 10-R2 strain, PR albino snails are thus potentially reduced in their natural capacity to mount cellular reactions against foreign materials.     

Chloroplasts in the epidermis of Sarracenia (the American pitcher plant) and their possible role in carnivory - An immunocytochemical approach

A photosynthetic apparatus is present in the epidermis of the bottom zone of the pitcher of Sarracenia purpurea L. ssp. purpurea. This has been demonstrated using conventional light and electron microscopy, as well as fluorescent and immunohistochemical techniques. Red intrinsic fluorescence by these chloroplasts indicates photochemical activity. Antibodies against the coupling factor of chloroplast ATPase and against the subunits of ribulose-bis-phosphate-carboxylase were bound to the epidermal chloroplasts. This has been visualized using a ferritin-isothiocyanate labeled second antibody. These results unequivocally prove the existence of the two main proteins which are associated with the photophosphorylation (membrane protein) and carbon dioxide fixation (stromal protein). The possible implication of this system to interrelationships between the carnivorous plant and aquatic insects inhabiting its leaves is discussed.     

Extracellular matrix (ECM) modulates the EGF-induced migration of liver epithelial cells in serum-free,hormone-supplemented medium

Summary The influence of the extracellular matrix (ECM) glycoproteins collagen, IV laminin (LN), and fibronectin (FN) on the in vitro migration of epithelial cells was studied using the ECM migration track method (4) with preparations immunostained for LN and FN. The locomotion of rat liver epithelial cells stimulated to migrate in serum-free medium by epidermal growth factor (EGF) in the presence of the protein per cm². Neither LN nor collagen IV decreased the number of migrating cells, indicating that the inhibition is a specific effect of fibronectin. The data also indicate that the FN-mediated inhibition of migration is an additional and not alternative mechanism to the well-established contact inhibition of locomotion (1) which also occurs in liver epithelial cell cultures. The system is being used for a further analysis of the factors that influence migration of normal and neoplastic epithelial cells and the biochemical mechanisms underlying the migration reaction. Editor’s Statement This paper describes new and heretofore neglected aspects of EGF and fibronectin action on the migratory behavior of cultured cells. Gordon H. Sato     

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.     

The Nuclear Envelope of Amoeba proteus During Mitosis as Studied With a Monoclonal Antibody Against a Membrane-Associated Protein

. The behavior of nuclear envelopes during mitosis in Amoeba proteus was studied by means of indirect immunofluo-rescence staining using a monoclonal antibody against a 220-kD membrane-associated protein of amoebae in conjunction with DAPI staining of chromatin. The antibody selectively recognized antigens on nuclear envelopes during interphase but did not react with the nuclear membranes during mitosis until after cytokinesis had been completed. Thus, it appeared that the membrane-associated protein reacting with the monoclonal antibody and normally present on the nuclear membranes was absent from fragmented nuclear membranes or nuclear membranes that were continuous but did not have the honey-comb lamina. The findings suggested that the 220-kD nuclear-membrane protein may be involved in the dissolution and reformation of the honey-comb lamina during mitosis in amoebae.