Dp44mT targets the AKT, TGF-ß and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells.

BACKGROUND: Effective treatment of prostate cancer should be based on targeting interactions between tumour cell signalling pathways and key converging downstream effectors. Here, we determined how the tumourigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumour-suppressive phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and transforming growth factor-ß (TGF-ß) pathways are integrated via the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumour agent, Dp44mT, may target these integrated pathways by increasing NDRG1 expression. METHODS: Protein expression in Dp44mT-treated normal human prostate epithelial cells and prostate cancer cells (PC-3, DU145) was assessed by western blotting. The role of NDRG1 was examined by transfection using an NDRG1 overexpression vector or shRNA. RESULTS: Dp44mT increased levels of tumour-suppressive PTEN, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras/MAPK signalling. Importantly, the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumour selectivity of these agents. Silencing NDRG1 expression increased phosphorylation of tumourigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, whereas NDRG1 overexpression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels. CONCLUSION: NDRG1 has an important role in mediating the tumour-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-ß and ERK pathways.

Oxytocin increases 5alpha-reductase activity of human prostate epithelial cells, but not stromal cells.

BACKGROUND: Oxytocin is known to modulate 5-alpha-reductase expression and has, therefore, been implicated in the etiology and novel pharmacological treatments of benign prostatic hyperplasia (BPH). These suggestions have been made in the absence of any direct evidence that oxytocin regulates expression or activity of 5-alpha-reductase isoenzymes in the human prostate. This study evaluated the effects of oxytocin on the activity and expression of 5-alpha-reductase isoenzymes I and II of human prostate stromal (PrSC; primary site of BPH development) and epithelial (PrEC) cells. METHODS: Cell cultures were incubated with oxytocin, or oxytocin plus a specific oxytocin antagonist for 24 hr, and conversion of (3)H-Testosterone to dihydrotestosterone used to estimate total 5-alpha-reductase activity and to determine activity of both type I and type II isoenzymes. Fully quantitative real-time RT-PCR determined levels of expression of both isoenzymes following treatments. RESULTS: Oxytocin significantly increased the total 5-alpha-reductase activity of PrEC but not of PrSC. 5-alpha-Reductase I gene expression and enzyme activity were also increased (P<0.05) in PrEC by oxytocin. Oxytocin significantly increased type II activity, but not expression, in PrEC. Oxytocin did not significantly affect 5-alpha-reductase activity or expression in PrSC. CONCLUSION: Both 5-alpha-reductase I and II are expressed in normal human prostate stromal and epithelial cells. Only 5-alpha-reductase isoenzymes of prostate epithelium are modulated by oxytocin.

Acute exposure of adult male rats to dietary phytoestrogens reduces fecundity and alters epididymal steroid hormone receptor expression.

Phytoestrogens are plant-derived compounds with oestrogenic activity. They are common in both human and animal diets, particularly through soy-based foods. This study assessed whether exposure of adult male rats to a high phytoestrogen diet for 3-25 days affected their fertility, and assessed possible mechanisms through which phytoestrogens may disrupt fertility. Adult males, fed a high phytoestrogen diet for 3 days, demonstrated significantly reduced fecundity. This effect was transient, with fecundity returning to control levels by day 12. The expression of oestrogen receptor-alpha and androgen receptor mRNA was increased in the initial segment of the epididymis, but decreased in the cauda epididymis following 3 days on the high phytoestrogen diet. Epididymal sperm counts cannot account for the reduction in fertility at day 3. However, lipid peroxidation of epididymal sperm was significantly increased in animals fed a high phytoestrogen diet for 3 days. Disruption of the steroid regulation of the epididymis by phytoestrogens may alter its function, resulting in decreased quality of sperm, and thereby reducing fecundity.

Regulation of 5alpha-reductase isoforms by oxytocin in the rat ventral prostate.

Oxytocin (OT) is present in the male reproductive tract, where it is known to modulate contractility, cell growth, and steroidogenesis. Little is known about how OT regulates these processes. This study describes the localization of OT receptor in the rat ventral prostate and investigates if OT regulates gene expression and/or activity of 5alpha-reductase isoforms I and II. The ventral prostates of adult male Wistar rats were collected following daily sc administration of saline (control), OT, a specific OT antagonist or both OT plus antagonist for 3 d. Expression of the OT receptor was identified in the ventral prostate by RT-PCR and Western blot, and confirmed to be a single active binding site by radioreceptor assay. Immunohistochemistry localized the receptor to the epithelium of prostatic acini and to the stromal tissue. Real-time RT-PCR determined that OT treatment significantly reduced expression of 5alpha-reductase I but significantly increased 5alpha-reductase II expression in the ventral prostate. Activity of both isoforms of 5alpha-reductase was significantly increased by OT, resulting in increased concentration of prostatic dihydrotestosterone. In conclusion, OT is involved in regulating conversion of testosterone to the biologically active dihydrotestosterone in the rat ventral prostate. It does so by differential regulation of 5alpha-reductase isoforms I and II.

Effects of steroids on oxytocin secretion by the human prostate in vitro.

Oxytocin (OT) concentrations are elevated in prostate tissue of patients with benign prostatic hyperplasia (BPH). Oxytocin specifically increases growth, 5 alpha-reductase activity and contractility in the prostate. In the rat prostatic OT concentrations are regulated by gonadal steroids, with androgens reducing but oestrogens increasing OT concentrations. The regulation of prostatic oxytocin in man is not understood. This study investigates the effects of gonadal steroids on oxytocin production by the human prostate. Primary explants (approx. 1 mm3) of prostate tissue from patients with BPH were incubated in Dulbecco's modified Eagle's media in the absence or presence of 10 nmol/L testosterone (T), 10 nmol/L dihydrotestosterone (DHT), T or DHT plus 100 nmol/L of the anti-androgen cyproterone acetate (CPA), 55 pmol/L diethylstilbestrol (DES), or DES plus DHT. The amount of oxytocin secreted into the media after 3 days was measured by radioimmunoassay. Testosterone and DHT significantly increased oxytocin concentrations secreted into the media from 0.86 +/- 0.11 ng/g of tissue (control) to 1.51 +/- 0.14 ng/g (p < 0.01) and 1.54 +/- 0.13 ng/g (p < 0.05), respectively. Incubation of tissue samples with CPA resulted in oxytocin concentrations similar to control levels. Treatment with DES caused a significant increase from 1.99 +/- 0.71 to 3.98 +/- 1.36 ng/g (p < 0.05). A similar increase was measured in media of tissue incubated in DES plus DHT (p < 0.001). The results demonstrate that, unlike the rat where androgens decrease oxytocin, in hyperplastic human prostate tissue both androgens and oestrogens increase oxytocin. This imbalance in the regulation of oxytocin may result in promoting prostatic overgrowth in the pathogenesis of BPH.

Oxytocin promotes spermiation and sperm transfer in the mouse.

Spermatogenesis is a complex process during which developing germ cells move from the base of the seminiferous tubule towards the lumen where they are shed. Studies in the rat suggest that seminiferous tubule contraction, induced by exogenous oxytocin, promotes spermiation. This study examines the role of testicular oxytocin in development of the testes, spermatogenesis and spermiation in the mouse. Groups of wild-type (WT) mice, oxytocin knockout mice (OTKO) deficient in testicular oxytocin and mice containing an oxytocin transgene (bOT4.2) that over express testicular oxytocin were killed between days 5 and 45 post partum. The testes and epididymides were removed weighed and prepared either for histological and morphometric study by light microscopy, for sperm counts (epididymis), or extracted for determination of oxytocin content (testis - day 45 only). Testicular oxytocin concentrations were significantly greater (p < 0.05) in bOT4.2 mice than in WT or OTKO mice. No differences in testicular and epididymal weight, or in diameter and area of seminiferous tubules between the mice genotypes were found at any given time. Germ cell development was similar in all genotypes and was comparable with previous studies. The timing of spermiation between the groups was significantly different (p < 0.001) with bOT4.2 < WT < OTKO and the appearance of epididymal sperm was significantly different (p < 0.05) with bOT4.2 < WT < OTKO. There were significant correlations between the percentage of tubules containing residual bodies and epididymal sperm count (p < 0.05) and between the percentage of animals containing residual bodies and the percentage of animals containing epididymal sperm (p < 0.01). These data suggest that in the mouse oxytocin, whilst not involved in germ cell development, is important in the process of spermiation and sperm transfer in the mouse.

Function and localization of oxytocin receptors in the reproductive tissue of rams.

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.

Oxytocin and vasopressin expression in the ovine testis and epididymis: changes with the onset of spermatogenesis.

Contractions of seminiferous tubules and epididymal duct walls promote spermiation and sperm transfer, and they are thought to be stimulated by the related peptides oxytocin and vasopressin. This study tested the hypothesis that if oxytocin and/or vasopressin play a physiological role in sperm shedding and transport, then local or circulating concentrations of these peptides would increase during puberty. Testes, epididymides, and trunk blood of sheep at stages during the first spermatogenic wave were collected, and radioimmunoassay measured significant increases in testicular and epididymal oxytocin during spermatogenesis. No changes were measured in circulating oxytocin or in local or circulating vasopressin. Localization and synthesis was investigated by immunohistochemistry and Western blot analysis employing antibodies recognizing epitopes of either oxytocin, oxytocin-associated neurophysin, vasopressin, or vasopressin-associated neurophysin. Marked expression of both oxytocin and its associated neurophysin in testicular Leydig and epididymal principal cells was seen, and weak neurophysin immunoreactivity was also identified in Sertoli cells. The intercellular distribution of oxytocin varied between regions of the epididymis, suggesting several roles for oxytocin. Vasopressin synthesis was not apparent in either tissue. These results confirm the presence and development of paracrine oxytocinergic systems in the ram testis and epididymis of ram during puberty while questioning the physiological importance of vasopressin.

Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

sodVIC is an alpha-COP-related gene which is essential for establishing and maintaining polarized growth in Aspergillus nidulans.

Strains of Aspergillus nidulans carrying the conditional-lethal mutation sodVIC1 (stabilization of disomy) are defective in nuclear division and hyphal extension. The mutation affects both the establishment and maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and preexisting hyphae stop growing upon upshift. The defect is reversible within the first 3-4 h at restrictive temperature but longer periods of incubation are lethal due to cell lysis and morphological abnormalities. There is no evidence for a specific cell cycle lesion, suggesting the existence of a feedback mechanism whereby hyphal extension is coordinated with nuclear partitioning. The sodVIC gene has been cloned from a chromosome VI-specific cosmid library and its product exhibits strong homology to the alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and higher organisms. Molecular disruption of the gene is lethal, indicating that SodVIC is essential for growth in A. nidulans.

Cloning, sequencing, temporal expression and tissue-specificity of two serine proteases from the midgut of the blood-feeding fly Stomoxys calcitrans.

Using highly degenerate, serine-protease-specific PCR primers on a midgut-specific cDNA library it was estimated that a minimum of 24 independent serine proteases were expressed in the midgut of Stomoxys calcitrans. The relative abundance of these 24 independent serine proteases has been estimated by restriction analysis of PCR products, showing that 69% fall into six almost equally abundant groups. Two highly abundant serine protease cDNAs (Ssp1 and Ssp2) were isolated and sequenced. They encode preproenzymes of 272 amino acids (Mr 28521) and 255 amino acids (Mr 27097) with putative signal peptides of 17 amino acids and 16 amino acids, putative activation peptides of 15 amino acids and 10 amino acids and mature enzymes of 239 amino acids (Mr 25322; pI 4.89) and 228 amino acids (Mr 24182; pI 7.59), respectively. Both deduced amino acid sequences contain the Asp/His/Ser catalytic triad and the highly conserved sequences surrounding it. Ssp2 also has the aspartate and two glycine residues in the specificity pocket, marking this as a typical trypsin. The positioning of the residues in the specificity pocket of Sspl is unusual; aspartate and glycine residues are present, which is typical of trypsin, but both are separated from surrounding conserved residues by additional amino acids; the second glycine found in the specificity pocket of trypsin is replaced by a serine, which is typical of chymotrypsin. Although a serine protease, the precise substrate specificity of Sspl remains to be determined. Northern analysis shows that both serine proteases are expressed constitutively with only a 20% change in the levels of expression of Ssp1 and Ssp2 through the digestive cycle, and that expression occurs predominantly in the opaque region of the midgut, the region responsible for secretion of digestive enzymes.

An in vitro system for the analysis of changes in depth distribution of diffusates in bacterial films.

No suitable technique exists which allows simultaneous analysis of time- and depth-dependent concentrations of all components of interest in single samples of intercellular fluid from dental plaque biofilms. We have developed an in vitro model which allows detailed study of these interactions by analysis of intercellular fluid and pH measurement at the film base. Compact, defined-depth films of Streptococcus mutans were formed in a micrometer-controlled, variable-depth well and bathed in synthetic saliva. Films exposed to synthetic saliva containing glucose for 2 min followed by clearance with glucose-free synthetic saliva gave typical 'Stephan-type' pH profiles. Intercellular fluid isolated from successive 200-microm-thick sections of 600-microm-deep films was analysed by ion chromatography. A concentration gradient of lactate, falling with depth, was measured. The experimental system described here, the first of its type to be described, can easily be used to analyse pH changes and the depth-dependent distribution of diffusates in a model bacterial film. Although this bacterial film is far removed from a natural biofilm, the apparatus has potential for the study of grown biofilms and is an important advance towards position-dependence analysis of diffusates in biofilms.

Mathematical model of beta-lactam penetration into a biofilm of Pseudomonas aeruginosa while undergoing simultaneous inactivation by released beta-lactamases.

We present a mathematical model that describes penetration of an antibacterial agent into a bacterial biofilm and, in particular, the penetration of a beta-lactam compound into a biofilm of Pseudomonas aeruginosa. As well as dealing with this penetration, and the consequent bacterial lysis, the model considered diffusion of the released beta-lactamases in the extracellular space and the consequent inactivation there of further incoming antibiotic; it also allowed for any chosen fraction of the total beta-lactamase to be permanently accessible to exogenous substrate. The modelling scheme was validated against analytical solutions under appropriately simplified conditions. Using published experimental data we show here that lysis of cells in the surface layers of a film could have an important protective effect on the viability of underlying bacteria, especially in thick biofilms.

Inhibition of acid production in Streptococcus mutans R9 by formic acid.

Both lactic and acetic acids cause mixed inhibition of acid production in mutans streptococci. This inhibition is partly irreversible due to cell death, an important factor when considering acidogenicity and aciduricity of these organisms, and their role in the caries process. Other monocarboxylic end-products may be present and may also be important inhibitors of acid production in dental plaque. This study considered the effects of varying concentrations of the end-product formic acid on acid production rates in Streptococcus mutans R9, measured using the pH-stat. Undissociated formic acid caused mixed inhibition with constants of Kiu (uncompetitive) of 6.07 +/- 1.27 mmol-1 and Kic (competitive) of 0.2 +/- 0.11 mmol l-1. Inhibition was found to be fully reversible, with no loss of cell viability. It is concluded that at those concentrations found in vivo, formate is not a significant inhibitor of acid production by S. mutans in dental plaque at any time, and is not important in determining the acidogenicity or aciduricity of this organism.

Inhibition of acid production in Streptococcus mutans R9: inhibition constants and reversibility.

End-product inhibition of acid production in Streptococcus mutans R9 was investigated by measuring effects of varying concentrations of H+ and of undissociated lactic or acetic acids on acid production rates in the pH stat. H+ caused purely uncompetitive inhibition (inhibition constant Kiu 0.018 mmol 1(-1). Lactic acid caused mixed inhibition with inhibition constants of Kiu 4.24 mmol 1(-1) and Kic 4.55 mmol 1(-1). Reversibility of inhibition by H+ showed only a statistically significant reduction only at pH < 4.5. Reversibility of inhibition by lactic and acetic acids decreased with increasing inhibitor concentration. In all cases, reversibility correlated with the extent to which viability was retained, suggesting that loss of reversibility was due to cell death. These results suggest that, after a low-pH episode in dental plaque, caused by fermentation of dietary carbohydrate, the ability of plaque organisms to produce acid in subsequent exposures to carbohydrate may be reduced.

Sequence analysis of the L1 metallo-beta-lactamase from Xanthomonas maltophilia.

The amino acid sequence deduced from the L1 beta-lactamase gene of Xanthomonas maltophilia shows a significant variation from that of the CphA and Blm metallo-beta-lactamases of Aeromonas hydrophila and Bacillus cereus, respectively. Whilst the N-terminus of the L1 protein shows some similarity, particularly at the histidine residues previously suggested as a zinc-binding motif, the C-terminus of the protein demonstrates very little similarity. Such differences amongst this group of enzymes would argue for at least three subclasses within the Group 3 beta-lactamases. However, in order to predict their phylogenetic ancestry more sequence data are required from other possible metallo-beta-lactamases.

A comparison of the multiple alleles of xylS carried by TOL plasmids pWW53 and pDK1 and its implications for their evolutionary relationship.

Both of the independently isolated TOL plasmids pWW53 and pDK1 contain multiple regions homologous to the xylS regulatory gene of the archetypal TOL plasmid pWW0. The three homologues on pWW53 vary in the extent of their homology to xylSpWW0, xylS1pWW53 is 99% identical to xylSpWW0 and is located relative to the single copy of xylRpWW53 in exactly the same way as xylS and xylR on pWW0. The DNA sequence of xylS3pWW53 is 87% identical to the xylSpWW0 sequence within the coding region but the non-coding DNA upstream is not homologous. There is a frame-shift change at the end of the coding region which causes the C terminus of XylS3pWW53 to be extended by an additional 10 amino acids relative to XylSpWW0. xylS2pWW53 is anomalous and appears to encode a truncated pseudogene lacking the first 525 bases found in the other xylS genes. Evidence is presented to show that both xylS1pWW53 and xylS3pWW53 act as regulators of meta pathway operons. Plasmid pDK1 carries two homologues of xylS. xylS1pDK1 is functional and is a hybrid gene: its 5' end and the upstream sequences are highly homologous to both xylS1pWW53 and xylSpWW0, whereas its 3' end is identical to xylS3pWW53. The sequence of xylS2pDK1 is identical to that of the anomalous truncated xylS2pWW53. Comparison of the organization and the restriction maps of the xyl catabolic operons on pDK1 and pWW53, together with the nucleotide sequences presented here, indicates that the catabolic DNA on pDK1 has derived from a replicon on which the xyl genes are organized similarly to pWW53 and that a genetic rearrangement has taken place involving a reciprocal recombination internal to two of its xylS homologues.

Construction of hybrid xylE genes between the two duplicate homologous genes from TOL plasmid pWW53: comparison of the kinetic properties of the gene products.

The two xylE genes for catechol 2,3-oxygenase, encoded by TOL plasmid pWW53, carry a common SalI restriction site within the reading frame. Each gene was cut at the SalI site and the 5' end of each gene spliced to the 3' end of the other to form hybrid genes, from both of which catalytically active catechol 2,3-oxygenase activities were expressed. The kinetic parameters were determined for the gene products of both the hybrid and the wild-type xylE genes with catechol, 3-methylcatechol and 4-methylcatechol as substrates. Comparison of the results suggested firstly, that the C-terminal regions of the enzymes determined both the binding and the catalytic specificity, and, secondly, that the N-terminal region of one of the enzymic gene products contained a secondary binding site which caused inhibition by excess substrate for methylcatechol substrates but not for catechol. One of the wild-type enzymes appeared to have an intrinsically higher activity for all three substrates than the other. This higher activity depended on the presence of both its C- and N-terminal regions, and in both hybrid enzymes, which contained only one of these regions, activity was significantly reduced.