Typing of Propionibacterium acnes: a review of methods and comparative analysis.

Propionibacterium acnes is a major commensal of the human skin. However, it is also the pathogen responsible for acne vulgaris and other diseases, such as medical-device infections. Strains of Propionibacterium acnes have long been classified into several different types. Recently, typing systems for this bacterium have taken on an increased importance as different types of P. acnes have been found to be associated with different disease states, including acne. Genetic approaches based on individual or multiple genes have classified P. acnes into types, which have been supported by the sequencing of nearly 100 P. acnes genomes. These types have distinct genetic, transcriptomic and proteomic differences. Additionally, they may have different immune response profiles. Taken together, these factors may account for the different disease associations of P. acnes types.

Transcription factor YY1: structure, function, and therapeutic implications in cancer biology.

The ubiquitous transcription factor Yin Yang 1 (YY1) is known to have a fundamental role in normal biologic processes such as embryogenesis, differentiation, replication, and cellular proliferation. YY1 exerts its effects on genes involved in these processes via its ability to initiate, activate, or repress transcription depending upon the context in which it binds. Mechanisms of action include direct activation or repression, indirect activation or repression via cofactor recruitment, or activation or repression by disruption of binding sites or conformational DNA changes. YY1 activity is regulated by transcription factors and cytoplasmic proteins that have been shown to abrogate or completely inhibit YY1-mediated activation or repression; however, these mechanisms have not yet been fully elucidated. Since expression and function of YY1 are known to be intimately associated with progression through phases of the cell cycle, the physiologic significance of YY1 activity has recently been applied to models of tumor biology. The majority of the data are consistent with the hypothesis that YY1 overexpression and/or activation is associated with unchecked cellular proliferation, resistance to apoptotic stimuli, tumorigenesis and metastatic potential. Studies involving hematopoetic tumors, epithelial-based tumors, endocrine organ malignancies, hepatocellular carcinoma, and retinoblastoma support this hypothesis. Molecular mechanisms that have been investigated include YY1-mediated downregulation of p53 activity, interference with poly-ADP-ribose polymerase, alteration in c-myc and nuclear factor-kappa B (NF-kappaB) expression, regulation of death genes and gene products, and differential YY1 binding in the presence of inflammatory mediators. Further, recent findings implicate YY1 in the regulation of tumor cell resistance to chemotherapeutics and immune-mediated apoptotic stimuli. Taken together, these findings provide strong support of the hypothesis that YY1, in addition to its regulatory roles in normal biologic processes, may possess the potential to act as an initiator of tumorigenesis and may thus serve as both a diagnostic and prognostic tumor marker; furthermore, it may provide an effective target for antitumor chemotherapy and/or immunotherapy.

Resveratrol and cancer: chemoprevention, apoptosis, and chemo-immunosensitizing activities.

The polyphenolic compound Resveratrol is a naturally occurring phytochemical and can be found in many plant species, including grapes, peanuts and various herbs. Several studies have established that Resveratrol can exert anti-oxidant and anti-inflammatory activities. It also has activity in the regulation of multiple cellular events associated with carcinogenesis. This review describes the general properties of Resveratrol including its relationship to estrogen, its effect on lipid metabolism, its cardiovascular effects, and its role on gene expression. Resveratrol has also been examined in several model systems for its potential effect against cancer. Its anti-cancer effects include its role as a chemopreventive agent, its ability to inhibit cell proliferation, its direct effect in cytotoxicity by induction of apoptosis and on its potential therapeutic effect in pre-clinical studies. In addition, Resveratrol has been shown to exert sensitization effects on cancer cells that will result in a synergistic cytotoxic activity when Resveratrol is used in combination with cytotoxic drugs in drug-resistant tumor cells. Clearly, the studies with Resveratrol provide support for the use of Resveratrol in human cancer chemoprevention and combination with chemotherapeutic drugs or cytotoxic factors in the treatment of drug refractory tumor cells.

Nitric oxide inhibits the transcription repressor Yin-Yang 1 binding activity at the silencer region of the Fas promoter: a pivotal role for nitric oxide in the up-regulation of Fas gene expression in human tumor cells.

NO has been increasingly implicated in control of the transcriptional machinery and serves as an intracellular second messenger to modify gene expression. We have demonstrated that NO up-regulated Fas receptor expression in ovarian carcinoma cell lines, albeit the mechanism involved is not known. Thus, we hypothesized that NO, directly or indirectly, may modify the transcriptional machinery that is responsible for the increased expression of the Fas gene. We examined the effect of NO on Fas gene expression using a Fas promoter-driven luciferase reporter system. Transient transfection of AD10 cells with pGL-3-FasP demonstrated that the IFN-gamma-dependent NO generation increases the trans-activation of the Fas promoter, and this increase was blocked by the NOS inhibitor (N(G)-monomethyl-L-arginine), but could be restored by the addition of the NO donor S-nitroso-N-acetylpenicillamine. Systematic deletion of the Fas promoter revealed that the functional region responsible for the NO-mediated effect was located at the silencer region, suggesting that NO may be responsible for the disruption of a repressor mechanism. We demonstrate that NO up-regulates the expression of the Fas receptor on AD10 cells via the specific inactivation of the transcription repressor yin-yang 1 DNA binding activity to the silencer region of the Fas promoter. These findings reveal a new mechanism of NO-mediated gene regulation by interfering with a repressor transcription factor at the silencer region of the Fas promoter.

Nitric oxide disrupts H2O2-dependent activation of nuclear factor kappa B. Role in sensitization of human tumor cells to tumor necrosis factor-alpha -induced cytotoxicity.

Tumor necrosis factor alpha (TNF-alpha) exerts its effect by two distinct signaling pathways. It can trigger cytotoxicity in sensitive target cells. TNF-alpha can also promote nuclear factor kappaB (NF-kappaB) activity and regulate the expression of genes that interfere with apoptosis and thus conferring resistance to several apoptotic stimuli. We have observed that interferon-gamma (IFN-gamma) sensitizes human ovarian carcinoma cell lines to TNF-alpha-mediated apoptosis and further, IFN-gamma induces the expression of the inducible nitric-oxide synthase (iNOS) and the generation of nitric oxide (NO). This study examines the role of NO in the sensitization of the ovarian carcinoma cell line AD10 to TNF-alpha-mediated cytotoxicity. Treatment of AD10 cells with the NOS inhibitor l-NMA blocked the IFN-gamma-dependent sensitization whereas NO donors (S-nitroso-N-acetylpenicillamine) sensitized these cells to TNF-alpha cytotoxicity. Analysis of the activation status of NF-kappaB upon treatment with NO donors confirmed the inhibitory role of NO on both the NF-kappaB DNA-binding property and its activation. Moreover, the inhibition of NF-kappaB nuclear translocation by NO donors directly correlated with the intracellular concentration of H(2)O(2) and was reversed by the addition of exogenous H(2)O(2). These findings show that NO might interfere with TNF-alpha-dependent NF-kappaB activation by interacting with O(2) and reducing the generation of H(2)O(2), a potent NF-kappaB activator. Therefore, NO-mediated disruption of NF-kappaB activation results in the removal of anti-apoptotic/resistance signals and sensitizes tumor cells to cytotoxic cytokines like TNF-alpha.

Nitric oxide sensitizes ovarian tumor cells to Fas-induced apoptosis.

Fas-mediated apoptosis represents one major mechanism by which tumor cells can be eliminated by activated cytotoxic immune lymphocytes. Previously, we have reported that interferon-gamma (IFN-gamma) sensitizes human ovarian carcinoma cell lines to Fas-mediated apoptosis. Furthermore, IFN-gamma, together with many other proinflammatory cytokines (TNF-alpha, IL-1beta, LPS, etc.), can stimulate the induction of inducible nitric oxide synthase (iNOS) and the generation of nitric oxide (NO). In this study, we examined whether nitric oxide is a mediator of IFN-gamma-induced sensitization of human ovarian carcinoma cell lines (A2780 and AD10) to Fas-mediated apoptosis and whether NO regulates the expression of the Fas receptor. Treatment of quiescent A2780 and AD10 ovarian carcinoma cells with IFN-gamma alone induced the expression of iNOS mRNA as examined by RT-PCR. There was accumulation of nitrite in the culture medium of IFN-gamma-treated cells, suggesting the generation of NOx. Like IFN-gamma, the use of exogenous sources of NO (S-nitroso-N-acetylpenicillamine (SNAP)) mimicked the sensitization of both cell lines to anti-Fas cytotoxic antibody (CH11) by IFN-gamma. Endogenously produced NO, by IFN-gamma pretreatment or exogenous nitrodonors, resulted in the upregulation of Fas receptor mRNA and protein expression. Blocking iNOS activity by NG-monomethyl-l-arginine (l-NMA) significantly reduced the sensitization, Fas mRNA, and protein expression observed with IFN-gamma pretreatment of the tumor cells. These findings demonstrate that sensitization of human ovarian carcinoma cell lines to Fas-mediated apoptosis by IFN-gamma can be due, in part, to the induction of iNOS and the subsequent upregulation of Fas gene expression by reactive nitrogen intermediates. Thus, the sensitivity of tumor cells to Fas-L-mediated cytotoxic immune lymphocytes can be regulated by the induction of NO or intermediates.

Transforming growth factor-beta1 (TGF-beta1) in penile and prostate growth in the rat during sexual maturation.

The goal of this study was to determine whether transforming growth factor-beta1 (TGF-beta1) may contribute to the arrest of penile growth and the down-regulation of androgen receptors (AR) that occur during sexual maturation in the rat penis. For this purpose, body, penis, and prostate weights were obtained from male rats of increasing ages, and penis and prostate TGF-beta1 concentrations were determined by a sandwich enzyme-linked immunosorbent assay. The cytosol fraction was obtained from the shafts and glandes of immature (19-day-old) and adult (90-day-old) rat penises, and ARs were measured by a western blot assay. The effect of exogenous TGF-beta1 on penile growth was examined in vivo in two groups of immature rats (21 and 27 days old) implanted with miniosmotic pumps delivering either human TGF-beta1 or vehicle only directly into the corpora cavernosa for 6 days. The penises, prostates, and testes were weighed, and the AR content was estimated by western blot. The growth rate of the penis declined after 8 weeks of age, whereas the ventral prostate growth rate increased until 14 weeks of age and then slowed down. The content of penile AR protein decreased seven-fold in the adult rats compared to the immature animals. Penile TGF-beta1 concentration increased nearly three-fold from the 19-day-old rats to a peak at 60 days of age and then decreased over the next 4 months to the initial levels. In contrast, TGF-beta1 concentration in the prostate was not significantly affected by age and remained below the lowest penile values in all age groups. Transforming growth factor-beta1 given locally to the penis reduced penile shaft weight by 38 and 22% in two groups of immature rats, while the weights of the penile glans, testis, and ventral prostate remained unaffected. Androgen receptor content was higher in the glans than in the shaft and was not changed by TGF-beta1 treatment. These results suggest that the increase of TGF-beta1 levels in the penis may reinforce growth arrest caused by the down-regulation of penile ARs, whereas the maintenance of a high content of ARs and a low TGF-beta1 concentration may allow prostate growth to continue.

Chemosensitization of human prostate carcinoma cell lines to anti-fas-mediated cytotoxicity and apoptosis.

Androgen ablation has been an effective treatment in patients with advanced prostate cancer. However, most treated patients develop hormonally resistant disease and do not respond to conventional chemotherapy. Immunotherapy against prostate cancer is an alternative approach in overcoming hormonal/drug-resistant prostate cancer. Cytotoxic immune lymphocytes kill target cells via the perforin/granzyme and the Fas-ligand (Fas-L) pathways. We hypothesize that tumor cells respond poorly to immunotherapy by developing resistance to killing by the Fas-L mechanism. This study investigated whether prostate tumor cells are sensitive to Fas-mediated killing. The human prostate carcinoma cell lines DU145, PC-3, and LnCAP were examined for their sensitivity to killing and apoptosis by the Fas-L agonist anti-Fas antibody and CTLs. All three lines moderately expressed the Fas antigen on the cell surface; however, all three lines were relatively resistant to cytotoxicity mediated by anti-Fas (CH-11) antibody. Pretreatment of DU145 and PC-3 with subtoxic concentrations of drugs followed by anti-Fas antibody resulted in synergistic cytotoxicity and apoptosis, whereas only an additive effect was obtained with LnCAP. Chemosensitization with drugs and anti-Fas was completely blocked by the addition of neutralizing anti-Fas antibody. The murine CTL hybridoma, PMMI, which kills only via the Fas-L pathway, was able to kill chemosensitized PC-3 and DU145 but not LnCAP cells. Furthermore, this cytotoxicity was blocked by anti-Fas neutralizing antibody. Chemosensitization of PC-3 and DU145 prostate tumor cells was not due to up-regulation of Fas-receptor antigen expression. Treatment of tumor cells with cisplatin did not down-regulate the antiapoptotic genes bcl-2, FAP-1, and c-myc. Further, there was no induction by cisplatin of Fas-L on the tumor cells, thus ruling out Fas/Fas-L-mediated autologous killing. These findings demonstrate that pretreatment of drug-resistant/CTL-resistant prostate DU145 and PC-3 tumor cells with subtoxic concentrations of certain chemotherapeutic drugs sensitizes the tumor cells to Fas-mediated cytotoxicity. These findings suggest that chemosensitization of tumor cells should optimize the response to immunotherapeutic interventions in the treatment of hormone-resistant/drug-resistant prostate cancer.

Cloning of rat and human inducible penile nitric oxide synthase. Application for gene therapy of erectile dysfunction.

Erectile dysfunction is mainly due to the inability of the cavernosal smooth muscle of the penis to undergo complete relaxation. In the aging rat model, erectile dysfunction is accompanied by a reduction of penile smooth muscle compliance and, in very old animals, by a decrease in penile nitric oxide synthase (NOS), which is responsible for the synthesis of the mediator of penile erection, nitric oxide (NO). We have investigated whether the stimulation of penile NOS expression by local induction or gene therapy can mitigate erectile dysfunction in the aged rat. A mix of iNOS (inducible NOS) inducers was continuously delivered to the penises of 5- ("adult"), 20- ("old"), and 30- ("very old") mo-old rats for 3-6 days, and the erectile response to electrical field stimulation of the cavernosal nerve was measured. The erectile dysfunction observed in old and very old rats as compared to adult animals was ameliorated by treatment with iNOS inducers. Penile iNOS was detectable in the penis of these rats by Western blot, NADPH diaphorase, and NOS activity assays. Inducible NOS was inducible in vitro in both rat and human corpora cavernosal tissue and in rat penile smooth muscle cells (RPSMC), as shown by Western blots. However, NO synthesis in cavernosal tissue upon iNOS protein induction remained low, indicating that the increased NOS levels were under physiological control. The iNOS cDNA was cloned from induced RPSMC mRNA and generated by reverse transcriptase polymerase chain reaction (RT-PCR) from induced human penile smooth muscle cells and corporal tissue. The coding regions from both the rat (RPiNOS) and human (HPiNOS) penile iNOS showed several amino acid differences from their analogous isoform in nonpenile tissues. RPiNOS cDNA injected into the penis mitigated the aging-associated erectile dysfunction. The iNOS construct was detected in cavernosal tissue by PCR, and its expression by RT-PCR and Western blots. These results open the way for the possible use of NOS isoforms in the management of erectile dysfunction.

Effect of long-term passive smoking on erectile function and penile nitric oxide synthase in the rat.

PURPOSE: Given that smoking is a risk factor for erectile dysfunction, this study aimed to determine, in a rat model, whether long-term exposure to cigarette smoke impairs nitric oxide (NO)-dependent erectile function and reduces penile nitric oxide synthase (NOS), and if these changes are accompanied with effects on the systemic blood pressure. MATERIALS AND METHODS: Adult (5 month) and old (20 month) rats were exposed to daily passive smoking for 8 wks. Three days after the conclusion of exposure, half of the animals were submitted to electrical field stimulation (EFS) of the cavernosal nerve and the maximum intracavernosal pressure (MIP) and mean arterial pressure (MAP) were determined and expressed as mm. Hg. On the other half of the animals, NOS activity in the penile cytosol was measured by the arginine/citrulline assay, and neuronal NOS (nNOS) and endothelial NOS (eNOS) contents were estimated by western blot and densitometry. RESULTS: When compared to controls, the smoking rats had a higher MAP in both the adult (115 vs 162) and old (113 vs 140) rats, but surprisingly the MIP also increased, from 78 to 111 (adult rats) and from 59 to 83 (old rats). Smoking reduced penile NOS activity by 73% (adult rats), and 62% (old rats), and nNOS content by 43% and 50%, respectively. In contrast, eNOS was not affected. Nitrite release, in vitro, by cavernosa slices or in rat penile smooth muscle cells (RPSMC) was not inhibited by nicotine or cotinine. CONCLUSION: These results indicate that chronic smoking in the rat leads to age-independent moderate hypertension and considerable decreases in penile NOS activity and nNOS content, that are not reflected in a reduction of the erectile response to EFS or accompanied by a decrease in penile eNOS.

Cloning of a novel neuronal nitric oxide synthase expressed in penis and lower urinary tract.

The neuronal nitric oxide synthase (nNOS) is the main NOS isoform in the urogenital tract catalyzing the synthesis of nitric oxide, the mediator of penile erection and presumably an important factor in the control of urinary voiding. We have cloned from the rat penile corpora cavernosa a cDNA coding for a novel nNOS differing from the cerebellar nNOS by the presence of a 102 nucleotides stretch and other features. This new species is the only nNOS mRNA expressed in the rat penis, urethra, prostate, and skeletal muscle, coexists with the cerebellar nNOS in the pelvic plexus and bladder, and is detectable in the cerebellum. The novel insert is present in human penile RNA and is transcribed from intron 16. The features and distribution of the penile nNOS suggest that it is may be regulated differentially from the cerebellar nNOS.

Reduction of penile nitric oxide synthase in diabetic BB/WORdp (type I) and BBZ/WORdp (type II) rats with erectile dysfunction.

Erectile dysfunction occurs frequently in human diabetes, and it is sometimes associated with hypogonadism. These conditions also develop in a model of insulin-dependent (type I) diabetes, the BB/WORdp (diabetic prone) rat but have not yet been investigated in the model of insulin-resistant (type II) diabetes, the BBZ/WOR rat. It is also unknown whether diabetes-related impotence is due to reduced levels of the mediator of penile erection, nitric oxide, caused by a decrease of nitric oxide synthase (NOS) in the penis. To clarify these questions, groups (n = 5-6) of diabetic BB/WORdp (insulin-maintained) and BBZ/WOR rats were age-matched with diabetic-resistant BB/WORdr and non-diabetic BB/WORdp rats and submitted to determinations of serum glucose, testosterone, and penile reflexes (cups and flips). Erectile dysfunction was found in all of type I and in most of type II diabetic animals (glycemias of 25.0 and 31.1 mM), at the selected mean ages of 310 and 180 days old, respectively. This was evidenced by over 95% decreases of erectile reflexes in both types of diabetes and was accompanied by 75% reduction of serum testosterone. Soluble NOS activity was measured in penile tissue from the diabetic rats with impaired erectile reflexes and in the corresponding controls, by the (3H)-L-arginine/citrulline conversion assay. The neuronal NOS isoform (nNOS) content was determined by a semiquantitative western blot assay. Both types of diabetes showed a marked decrease of penile NOS activity (74 and 55%, respectively), and a lower reduction of penile nNOS content (47 and 33%, respectively). No endogenous NOS inhibitor was detected in the diabetic type I penile cytosol by cross-mixing NOS activity assays. Our data support a common etiology for erectile dysfunction present in rats with types I and II diabetes mellitus and suggest that the etiology is related to a decrease of penile NOS derived in part from serum androgen deficiency.

Restoration of normal adult penile erectile response in aged rats by long-term treatment with androgens.

In order to determine whether the aging-related impairment of penile erection can be corrected by exogenous androgens, 20-mo-old ("old") rats received implants of Silastic tubing containing either testosterone (T) or dihydrotestosterone (DHT). Untreated aged-matched rats were used as reference controls, and values were compared with those obtained previously for 5-mo old ("adult") rats. After 45 days, groups of six rats were submitted to electrical field stimulation of the cavernosal nerve (EFS), and the intracavernosal pressure and mean arterial pressure (MAP) were recorded. Compared to the untreated old rats, the old animals receiving either T or DHT showed a similar and significant (p < 0.01) increase (56%) in the maximal intracavernosal pressure (MIP) to a level slightly above that of the untreated adult rats. The response was sensitive to the nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester (L-NAME). The MIP:MAP ratio in the treated animals was nearly double that found in the old untreated controls. Penile NOS activity was measured by the [3H]L-arginine-citrulline conversion, and neuronal NOS (nNOS) levels were estimated by a semiquantitative Western blot assay with antibodies against human nNOS. No significant variations were observed in either NOS levels or NOS activity between the treated and untreated old rats. These results suggest that aging-related erectile dysfunction in the intact rat may be responsive to androgen therapy and that this correction is not associated with an increase in the basal levels of penile NOS, in contrast to what occurs in castrated rats.

Effect of aging on nitric oxide-mediated penile erection in rats.

Aging is an important risk factor for impotence in men. Because nitric oxide (NO) appears to be the mediator of corpora cavernosal smooth muscle relaxation, we have examined in 5-, 20-, and 30-mo-old rats, designated "adult," "old," and "senescent," respectively, whether aging causes a decrease of erectile response that may correlate with lower NO synthase (NOS) in the penis. Electric field stimulation (EFS) of the cavernosal nerve showed that the maximum intracavernosal pressure (MIP) declined in the old and senescent rats to 80 and 51% of the adult value, respectively. A low systemic dose of the NOS inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME; 2 mg/kg), reduced the MIP by only 38% in the adult rats but decreased it in the old and senescent rats by 72 and 80%, respectively. In the absence of EFS, intracavernosal papaverine (phosphodiesterase inhibitor), or nitroglycerin (NO donor), caused a lower erectile response in the old and senescent rats compared with the adult animals (MIP: 41 and 14%, respectively; duration of the erection 46 and 21%, respectively). Tissue sections from old and senescent penises showed increasing degrees of sclerotic degeneration. In comparison with the adult rats, the penile soluble NOS activity per gram of tissue that is sensitive to L-NAME decreased significantly by 63% in the senescent rats but was elevated in the old rats. These results indicate that aging causes an erectile failure due to factors initially independent from an impairment of penile NO synthesis but which are compounded in the very old rats by the decrease of penile NOS activity.