Phage display selection of peptides that affect prostate carcinoma cells attachment and invasion.

BACKGROUND: Prostate cancer-specific proteins must be identified to serve as diagnostic and prognostic markers. Cell surface proteins are especially important, because they have potential utility as diagnostic markers and therapeutic targets. Identification of ligands for these proteins will allow use of these ligands as diagnostic and therapeutic tools and permit the investigation of receptor function. We performed a search for peptide ligands to prostate cancer cell-specific receptors. METHODS: Peptide phage display library was used to isolate specific ligands to LNCaP prostate carcinoma cells receptors. Selected phage and cognate peptides were investigated for their cancer-related functions, such as the ability to interfere with cell adhesion, spreading, motility, and invasion. RESULTS: Phage designated pg35, blocked spreading of LNCaP cells and their derivatives C4-2 and C4-2b. Cognate peptide did not inhibit spreading, but incubation of C4-2 and C4-2b cells with cognate peptide increased their affinity for endothelial cells and invasiveness. In addition, the peptide activates matrix metalloproteinase (MMP)-2 and 9 in C4-2 and C4-2b cells. CONCLUSIONS: These results indicate that identified ligands may play a role in tumorigenicity and metastatic transformation of prostate cancer. To our knowledge, this is the first identification of a functional cancer-specific peptide ligand using the phage display approach.

[The enzymatic activity of the antioxidant system in the ontogeny of mutant norflurazone-tolerant Arabidopsis thaliana]. / Izuchenie aktivnosti fermentov antioksidantnoi sistemy v ontogeneze mutantov Arabinopsis thaliana, tolerantnykh k norflurazonu.

The activities of superoxide dismutase and guaiacol-dependent peroxidase were studied in the ontogenesis of recessive homozygous mutants of Arabidopsis thaliana Heynh. le-2 and nfz24, which are characterized by two- to threefold increases in tolerance to the herbicide norflurazone. The mutants le-2 and nfz24 differed from the initial race Dijon in some phenotypic features, duration of ontogenetic stages, and dynamics of the superoxide dismutase and peroxidase activities in ontogenesis. A single treatment of plants with norflurazone induced an accelerated increase in the level of both enzymes in the mutants as compared to the wild type plants. Under the conditions of multiple treatment with norflurazone, the mutants le-2 and nfz24 displayed a higher tolerance to the bleaching effect of the herbicide and were characterized by a higher level of superoxide dismutase. The data obtained suggest that the superoxide dismutase and peroxidase activities are controlled by both ontogenetic factors and stress signals. Mutations in the lines le-2 and nfz24 increase sensitivity to a stress signal or increase efficiency of an adaptive response due to long-term maintenance of a high level of the antioxidant enzymes under the conditions of stress.

[Experience with glyciphonic ointment for malignant and precancerous lesions of the skin]. / Opyt primeneniia glitsifonovoi mazi pri rake i predrakovykh zabolevaniiakh kozhi.

Skin was smeared with glyciphonic ointment containing 30% of methylphosphonic diglycidyl ether (Tatkhimfarmpreparaty Company) in 495 patients with histologically confirmed basalioma and 36 patients with senile keratosis. During daily application necrotic tissue was removed. Considerable decomposition of tumor occurred on day 6 or later. It took the wound 9-12, seldom, 15 days to heal, with scars forming on days 15-20. Stable cure was registered (5-7 yrs) in 492 patients with skin basalioma and all cases of senile keratosis. No changes in peripheral blood count or any skin-resorption side-effects were recorded. Several patients suffered hyperemia, edema, itching or local pain.

Reduced tolerance to acute renal ischemia in mice with a targeted disruption of the osteopontin gene.

BACKGROUND: Mice with a targeted disruption of the osteopontin gene through homologous recombination in embryonic stem cells have recently been generated and shown to be characterized by unaltered fertility and normal embryonic and postnatal development, including renal development, but altered osteoclastogenesis from spleen progenitors. The lack of detectable pathological manifestations in kidneys of mice with the targeted disruption of the osteopontin gene (opn -/-) makes them an excellent model for studies of pathophysiological processes that are thought to be accompanied by changes in renal osteopontin expression. It has previously been suggested that osteopontin may play an important role in the pathophysiology of acute renal failure, thus prompting this study. METHODS: Wild-type and opn -/- mice were subjected to 30 minutes of renal ischemia and were studied 24 hours later. RESULTS: Control opn +/+ mice showed a significant retention of blood urea nitrogen and creatinine, which is indicative of the development of ischemic acute renal dysfunction. This was accompanied by a 2.7-fold increase in the immunodetectable osteopontin compared with sham-operated control. Animals with the disrupted osteopontin gene exhibited ischemia-induced renal dysfunction, which was twice as pronounced as that observed in mice with the intact osteopontin response to stress. In addition, the structural damage to the ischemic kidneys obtained from opn -/- mice was more pronounced than that observed in similarly treated wild-type mice. This was associated with the augmented expression of inducible nitric oxide synthase and the prevalence of nitrotyrosine residues in kidneys from opn -/- mice versus wild-type counterparts. In vitro studies with proximal tubular cells subjected to hypoxia in the presence of OPN, but not OPN with deleted arginine-glycine-aspartic acid (RGD) domain, resulted in cytoprotection. CONCLUSIONS: The comparative analysis of functional and morphological sequelae of acute renal ischemia in opn +/+ and opn -/- mice provides strong evidence of renoprotective action of osteopontin in acute ischemia.

RGD-recognizing integrins mediate interactions of human prostate carcinoma cells with endothelial cells in vitro.

BACKGROUND: Interactions of cancer cells with endothelium are a crucial step in metastatic invasion. RGD-recognizing integrins play a definitive role in these interactions. METHODS: Fluorescence-activated cell sorting (FACS) analysis of RGD-sensitive integrins in prostate epithelial cells was performed. Attachment inhibition assay was used to characterize functionality of particular integrins. Potential partners for RGD-binding integrins in human umbilical vein endothelial cells (HUVEC) were identified by Western blotting and attachment inhibition assay. To determine the RGD-flanking amino acids optimal for interactions with prostate cell integrins, these cells were biopanned with a phage library. RESULTS: Different expressions of RGD-recognizing integrins and distinctions in RGD-dependent adhesion of nonmalignant and cancer cells were observed. Cancer but not control cells were detached from culture plastic by incubation with RGD peptide. Adhesion of carcinoma cells to HUVEC was RGD-sensitive, in contrast to nonmalignant cells. Antibodies against alpha3, alpha5, beta1, and alpha(v)beta3 inhibited interactions of carcinoma cells with HUVEC. Potential ligands for alpha5beta1, alpha3beta1, and alphaVbeta3 integrins, fibronectin, and vitronectin, were detected on the HUVEC surface. Several phages which preferentially bound to the surface of particular prostate cells were selected. CONCLUSIONS: Interactions of prostate carcinoma with endothelium are mediated in part via alpha5beta1, alpha3beta1, and alpha(v)beta3 integrins. Because these interactions are RGD-sensitive, synthetic RGD peptides with optimized flanking amino acids can potentially be used as antimetastatic agents.

Posttranscriptional regulation by Rev protein of human immunodeficiency virus type 1 results in nonrandom nuclear localization of gag mRNA.

The expression of the human immunodeficiency virus type 1 mRNAs containing the Rev-responsive element is regulated at the posttranscriptional level by the viral Rev protein. Rev increases the nucleocytoplasmic export of these mRNAs, leading to high expression. Using in situ hybridization and electron microscopy, we investigated the localization of a subgenomic gag mRNA in the absence and presence of Rev. In addition to the previously shown cytoplasmic accumulation of the Rev-dependent mRNA, we observed that in the presence of Rev the nuclear gag mRNA accumulates nonrandomly and forms specific localization patterns at the nuclear membrane and in the nucleoplasm. Cellular mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase were not found to form such patterns. These data suggest that Rev leads the gag mRNA to specific subnuclear locations, which further supports the transport function of Rev.

Endocytosis of gastrin in cancer cells expressing gastrin/CCK-B receptor.

Endocytosis of gastrin was studied in a number of gastrin-receptor-expressing cell lines by confocal laser scanning microscopy (CLSM) with the aid of a biologically active fluorescent derivative, rhodamine green heptagastrin. Rapid clustering (within 4-7 min) and internalization of fluorescent ligand upon binding at room temperature and 37 degrees C were observed in the rat pancreatic acinar carcinoma cell line AR42J, human gastric carcinomas AGS-P and SIIA, human colon carcinomas HCT116 and HT29, and in NIH/3T3 cells transfected with human and rat gastrin/cholecystokinin-B receptor cDNA. Internalization was inhibited by hypertonic medium. Fluorescent heptagastrin and transferrin colocalized in the same endocytic vesicles at different stages of internalization suggesting that endocytosis occurred predominantly through a clathrin-dependent mechanism. At 37 degrees C partial colocalization with the lysosomal marker neutral red was detected by CLSM, implying that internalized gastrin accumulated in the lysosomes. Immunoelectron microscopy studies with antibodies against gastrin revealed the presence of the internalized hormone in multivesicular vesicles and endosomes. Almost no hormone was detected in lysosomes with the antibodies to gastrin, suggesting that the degradation of the peptide is rapid in those vesicles. Continuous accumulation of fluorescent label was observed by CLSM in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the gastrin receptor is recycled back to the cell membrane after hormone delivery to intracellular compartments. An estimated average recycling time for the receptor molecules was 1 h in NIH/3T3 cells.

Numerous cell targets for gastrin in the guinea pig stomach revealed by gastrin/CCKB receptor localization.

The localization of the gastrin/CCKB receptor (GR) has recently become a subject of debate, especially since the publication of evidence for its presence in an unsuspected location, namely in the lamina propria, the submucosal layer of the stomach lining. Knowledge of the receptor localization is important because of the critical role of gastrin secretion and its trophic effects on the gastric epithelium. The present study, which utilizes immunohistochemistry and electron microscopy as primary tools, provides unequivocal data concerning the localization of GR in the guinea pig stomach. GR is expressed in parietal cells, on chief cells, and in previously unreported endocrine cells of the stomach. It is not found in the lamina propria. The predominant localization of the receptor in the endocrine cells is on the membranes of cytoplasmic electron-dense secretory granules. The positioning of these cells in the gastric glands suggests that they may be involved in the uptake of gastrin from the circulation. The distribution of GR implies that it may be involved in the regulation of various processes and may mediate various effects of gastrin in the stomach.

Protein synthesis is required for laminin-induced expression of the 67-kDa laminin receptor and its 37-kDa precursor.

The high affinity 67-kDa laminin receptor (67LR) is highly expressed in metastatically active human cancers. A 37-kDa polypeptide has been identified as its precursor (37LRP). Antibodies raised against 37LRP-derived synthetic peptides were used in immunogold electron microscopy and immunoblot studies to assess the effect of laminin on expression of the 67LR and the 37LRP. Laminin (15 micrograms/ml) treatment of suspended A2058 human melanoma cells doubled the expression of both 37LRP and the 67LR. Fibronectin had no effect. There was no effect of laminin on the expression of actin or galectin-3. Cycloheximide treatment of cells prior to laminin abrogated its inducible effect. The results suggest that binding of laminin by cell surface laminin receptors induces synthesis of the 37LRP and mature 67LR, with a consequent delivery to the cell surface of more laminin binding proteins for potentiated attachment of the melanoma cell to the basement membrane during invasion and metastasis.

Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids.

We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.

[Ultrastructure of the apical plasma membrane of the granular cells in the frog bladder during cobalt-ion decrease in the vasopressin effect]. / Ul'trastruktura apikal'noi plazmaticheskoi membrany granuliarnykh kletok mochevogo puzyria liagushki pri snizhenii éffekta vazopressina ionami kobal'ta.

Simultaneous studies were performed on changes in water permeability and on the ultrastructural organization of the frog urinary bladder epithelium in the presence of Co-ions under vasopressin-stimulated water flow. A possible inhibition of the vasopressin-stimulated water flows by Co-ions is supposed from the extracellular surface of the apical membrane of granular cells responsible for water permeability of this epithelium. Using the freeze-fracture technique for studying the apical membrane ultrastructure, it was shown that with the maximum water flow the square occupied by intramembrane particle aggregates was as much as 1.8% of the total square of membranes, to reduce to 0.3% with the smaller water flow, the average sizes of aggregates being 0.35 mkm and 0.08 mkm in both these cases, respectively. Application of 1 x 10(-3)-1 x 10(-4) M CoCl2 from the mucose part inhibits the vasopressin-stimulated water flow. In this case no aggregates are actually seen on the P-face of the apical membrane, the number of intramembrane particles of the E-face being similar to that when the water permeability was originally low. It is concluded that Co-ion may influence the structure and function of the apical plasma membrane from its extracellular surface.

[Ultrastructural changes in the plasma membrane of A-431 cells exposed to epidermal growth factor]. / Izmenenie ul'trastruktury plazmaticheskoi membrany kletok A-431 pri deistvii épidermal'nogo faktora rosta.

The plasma membrane ultrastructural changes after the action of epidermal growth factor were studied in A-431 cells using freeze-fracture methods. The incubation with EGF (100 ng/ml, 0 degree C, 60 min) led to a decrease in density of intramembrane particles on the P surface of ventral cell membrane, while the number of coated pits increased there. The revealed effects of EGF may be related to direct consequences of EGF-receptor complex formation, because all the temperature dependent steps of its processing were blocked. The data obtained testify to an active involvement of the membrane ventral surface in the formation of cell response towards growth factors.

[Structural and functional characteristics of the cellular reaction of the bladder epithelium in the frog to calcium extraction from the apical and basolateral membranes]. / Strukturno-funktsional'nye osobennosti reaktsii kletok épiteliia mochevogo puzyria liagushki na izvlechenie kal'tsiia iz apikal'noi i bazolateral'noi membran.

Extraction of Ca++ ions from cells of the frog urinary bladder serosa side is followed by an increase in the bladder wall permeability for water and inulin. Ultrastructural changes were observed, such as destruction of cell junctions, swelling of the cell and their organelles, reconstruction of the cytoskeleton elements. The free calcium Ringer solution injected in the bladder lumen does not change the permeability of the wall for water and sodium ions. In this case the cell response to the antidiuretic hormone decreases; the ultrastructure of cells and intercellular junctions is not disturbed; the distribution of intramembrane particles on the P- and E-faces of the apical membrane is normal. The above results indicate that there are qualitative differences in the cell response towards the extraction of Ca++-ions between the serosal and mucosal membranes. This also suggests that on the external surface of the apical membrane Ca++ ions may play a very important role in redistribution of intramembrane particles under the action of the antidiuretic hormone.

[Glucose metabolism in isolated bacteroids of lupine nodules]. / Obmen gliukozy v izolirovannykh bakteroidakh kluben'kov liupina.

The ability of the bacteroids Rhizobium lupini isolated from the nodules at the stage of active nitrogen fixation to consume [1-14C]- and [2-14C] glucose was studied. 5 min after glucose addition to the cell suspension the label is rapidly incorporated into the organic acids, amino acids and sugars, which is indicative of glucose penetration inside the bacteroids and of a high rate of its catabolism. The rapid incorporation of the label into all Krebs cycle metabolites and the activities of citrate (si)-synthase, isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, which are positively correlated with the rate of the nitrogen fixation process in the course of plant vegetation provide evidence for the occurrence of the tricarboxylic acid cycle in the bacteroids.

[Paraffin oxidizing system of the yeast Candida guilliermondii]. / Parafinokisliaiushchaia sistema drozhzhei Canidia quilliermondii.

It has been demonstrated that the enzyme system of Candida guilliermondii responsible for hydrocarbon oxidation involves NADPH-cytochrome c-reductase (EC 1623) and cytochrome P-450. The system is located in the microsomal fraction. Cytochrome P-450 synthesis is induced by hexadecane occurring in the medium. The cellular content of cytochrome P-450 varies in the course of the culture growth. There is a correlation between the cellular content of cytochrome P-450 and the synthesis of primary products of hexadecane oxidation.