Extracellular alpha 6 integrin cleavage by urokinase-type plasminogen activator in human prostate cancer

During human prostate cancer progression, the integrin alpha6beta1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. We previously identified a novel structural variant of the alpha6 integrin called alpha6p. This variant was produced on the cell surface and was missing the beta-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of alpha6p. We also showed that addition of uPA in the culture media of cells that do not produce alpha6p, resulted in a dose-dependent alpha6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using alpha2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly. Further, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not alpha2-antiplasmin. Finally, the alpha6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the beta-barrel ligand-binding domain of the integrin while preserving its heterodimer association.     

Androgen control of cell proliferation and cytoskeletal reorganization in human fibrosarcoma cells: role of RhoB signaling

We recently generated an HT-1080-derived cell line called HT-AR1 that responds to dihydrotestosterone (DHT) treatment by undergoing cell growth arrest in association with cytoskeletal reorganization and induction of neuroendocrine-like cell differentiation. In this report, we show that DHT induces a dose-dependent increase in G0/G1 growth-arrested cells using physiological levels of hormone. The arrested cells increase in cell size and contain a dramatic redistribution of desmoplakin, keratin 5, and chromogranin A proteins. DHT-induced cytoskeletal changes were also apparent from time lapse video microscopy that showed that androgen treatment resulted in the rapid appearance of neuronal-like membrane extensions. Expression profiling analysis using RNA isolated from DHT-treated HT-AR1 cells revealed that androgen receptor activation leads to the coordinate expression of numerous cell signaling genes including RhoB, PTGF-beta, caveolin-2, Egr-1, myosin 1B, and EHM2. Because RhoB has been shown to have a role in tumor suppression and neuronal differentiation in other cell types, we investigated RhoB signaling functions in the HT-AR1 steroid response. We found that steroid induction of RhoB was DHT-specific and that newly synthesized RhoB protein was post-translationally modified and localized to endocytic vesicles. Moreover, treatment with a farnesyl transferase inhibitor reduced DHT-dependent growth arrest, suggesting that prenylated RhoB might function to inhibit HT-AR1 cell proliferation. This was directly shown by transfecting HT-AR1 cells with RhoB coding sequences containing activating or dominant negative mutations.     

Integrin clipping: A novel adhesion switch?

During human prostate cancer progression, the majority of normally expressed integrins are suppressed with the exception of the alpha6, alpha3, and beta1 integrins. We have shown that in prostate cancer, the alpha6 integrin is found paired with the beta1 integrin and that a novel form of the alpha6 integrin that lacks a large portion of the extracellular domain (alpha6p) exists. The alpha6pbeta1 integrin is found in human prostate cancer tissue specimens as well as tissue culture cell lines and is formed on the cell surface. This review discusses the mechanism of alpha6pbeta1 production and the potential functions of this integrin variant. Our current working model predicts that the alpha6pbeta1 integrin maintains the intracellular cytoskeletal connections associated with the heterodimer while allowing for an alteration in cell adhesion. The mechanism provides a selective advantage for cancer cell metastasis.     

Biochemical comparisons of habituated and non-habituated callus lines of Glycine max (L.) cv. Acme

A cytokinin habituated non-organogenic callus line of Glycine max L. cv. Acme, was isolated. Cytokinins were detected in both non- and habituated calli. HPLC analysis indicated that the highest biological activity was associated with compounds that cochromatographed with zeatin and derivatives thereof. Cytokinin-like activity in the habituated callus was higher than in the non-habituated callus. No difference was observed in the ability of the two callus types to metabolise trans-zeatin. The habituated callus however, metabolised N⁶(²-isopentenyl)adenine (iP)at a slower rate than the non-habituated line. The regulation of proline and putrescine levels during the growth cycle was similar in both callus types, although less proline was present in the habituated than the non-habituated callus. In both callus types a decrease in putrescine levels was accompanied by an increase in the proline levels on day 21 of the growth cycle. Neither spermine nor spermidine were detected in either the habituated or non-habituated lines. A comparison of the total RNA levels of the habituated and non-habituated material revealed higher RNA levels in the non-habituated than the habituated line. Two-dimensional gel electrophoresis indicated the presence of 3 polypeptides in the habituated callus on day 13 of the growth cycle. These were not detected in the non-habituated callus, of similar age.Abbreviations Ade Adenine - DTT 1,4-dithiothreitol - IEF isoelectrofocussing - iP N⁶(²-isopentenyl)adenine - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulphate     

Intracellular Localization of the Neurotoxin 2,4-Diaminobutyric Acid in Lathyrus sylvestris L. Leaf Tissue

The intracellular distribution of the neurotoxin 2,4-diaminobutyric acid (DABA) in mature leaves of the perennial legume Lathyrus sylvestris L. var `Lathco' (flatpea) was determined using subcellular fractions from mesophyll protoplasts. Chloroplasts contained about 15% of the cellular DABA. At least 75% of the DABA was vacuolar, based on the assumptions that each protoplast contained a single vacuole and that acid phosphatase occurred exclusively in the vacuole. DABA was not detectable in peroxisomal and mitochondrial fractions. Because the vacuole is not a major site of amino acid synthesis, this distribution implicates synthesis of DABA within chloroplasts with subsequent transport to and storage within the vacuoles of the mesophyll cells.     

Site-specific mutagenesis of potato spindle tuber viroid cDNA:

Summary The infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered wild type sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.     

Stimulation of the de novo pathway for the biosynthesis of platelet-activating factor (PAF) via cytidylyltransferase activation in cells with minimal endogenous PAF production

Treatment of Ehrlich ascites cells with 2 mM oleic acid causes a greater than 10-fold increase in the formation of platelet-activating factor (PAF; 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) from the de novo precursor of PAF, 1-[3H]alkyl-2-acetyl-sn-glycerol. Under these conditions, CTP:phosphocholine cytidylyltransferase activity, which is known to catalyze the rate-limiting step in phosphatidylcholine biosynthesis, was stimulated 32% (p less than 0.001) over control cells. Surprisingly, the dithiothreitol-insensitive choline-phosphotransferase activity, which catalyzes the final step in PAF biosynthesis, was reduced approximately 95% in membranes isolated from cells that were pre-treated with 2 mM oleic acid. However, calculations of product formation at this reduced cholinephosphotransferase activity revealed that it was still sufficient to accommodate the increased synthesis of PAF observed in the intact oleic acid-treated cells. Kinetic studies and experiments done with cells treated with phenylmethylsulfonyl fluoride (an acetylhydrolase inhibitor) indicate the various metabolic products formed are derived through the following sequence of reactions: 1-alkyl-2-acetyl-sn-glycerol----1-alkyl-2-acetyl-sn-glycero-3- phosphocholine----1-alkyl-2-lyso-sn-glycero-3-phosphocholine----1-alkyl- 2(long-chain) acyl-sn-glycero-3-phosphocholine. These results indicate PAF is the source of alkylacylglycerophosphocholine through the action of an acetylhydrolase and a transacylase as shown in other cell systems. The relative amounts of PAF, lyso-PAF, and alkylacylglycerophosphocholine produced after treatment of the cells with oleic acid in the absence of the phenylmethylsulfonyl fluoride inhibitor indicate that the acylation rate for lyso-PAF is considerably slower (i.e. rate-limiting) than the deacetylation of PAF by acetylhydrolase. We further conclude that the final step in the de novo pathway for PAF biosynthesis is under the direct control of CTP:phosphocholine cytidylyltransferase, which emphasizes the importance of this regulatory (rate-limiting) step in the biosynthesis of both phosphatidylcholine and PAF.     

Education Program Evaluation at Multiple Primate Sanctuaries in Equatorial Africa

The past decade has witnessed a shift in the role of primate sanctuaries in Africa from warehouses of salvaged animals to proactive conservation organizations. As part of this new role, the Pan African Sanctuary Alliance (PASA) has conducted annual training workshops for educators from member sanctuaries. These workshops have included content on program development, interpretive techniques, and evaluation techniques. Here we use meta-analytic techniques to examine data from the resulting education evaluation programs for trends in knowledge acquisition across PASA sanctuaries. Question content strongly impacted the evaluation results of the conservation education program. In addition, effect size and final performance scores were independent of one another and provide different perspectives as to the effectiveness of a conservation education program. These results highlight the importance of using both final performance scores and effect size measures in conservation education program evaluation. They also underscore the importance of evaluating knowledge transfer as part of the larger picture of evaluating pro-environmental behavior change and conservation impact.