Silencing of integrated human papillomavirus type 18 oncogene transcription in cells expressing SerpinB2

The serine protease inhibitor SerpinB2 (PAI-2), a major product of differentiating squamous epithelial cells, has recently been shown to bind and protect the retinoblastoma protein (Rb) from degradation. In human papillomavirus type 18 (HPV-18) -transformed epithelial cells the expression of the E6 and E7 oncoproteins is controlled by the HPV-18 upstream regulatory region (URR). Here we illustrate that PAI-2 expression in the HPV-18-transformed cervical carcinoma line HeLa resulted in the restoration of Rb expression, which led to the functional silencing of transcription from the HPV-18 URR. This caused loss of E7 protein expression and restoration of multiple E6- and E7-targeted host proteins, including p53, c-Myc, and c-Jun. Rb expression emerged as sufficient for the transcriptional repression of the URR, with repression mediated via the C/EB beta-YY1 binding site (URR 7709 to 7719). In contrast to HeLa cells, where the C/EBP beta-YY1 dimer binds this site, in PAI-2- and/or Rb-expressing cells the site was occupied by the dominant-negative C/EBP beta isoform liver-enriched transcriptional inhibitory protein (LIP). PAI-2 expression thus has a potent suppressive effect on HPV-18 oncogene transcription mediated by Rb and LIP, a finding with potential implications for prognosis and treatment of HPV-transformed lesions.

Is cannabis use a contributory cause of psychosis?

Objective: To assess whether cannabis use in adolescence and young adulthood is a contributory cause of schizophreniform psychosis in that it may precipitate psychosis in vulnerable individuals. Method: We reviewed longitudinal studies of adolescents and young adults that examined the relations between self-reported cannabis use and the risk of diagnosis with a psychosis or of reporting psychotic symptoms. We also reviewed studies that controlled for potential confounders, such as other forms of drug use and personal characteristics that predict an increased risk of psychosis. We assessed evidence for the biological plausibility of a contributory causal relation. Results: Evidence from 6 longitudinal studies in 5 countries shows that regular cannabis use predicts an increased risk of a schizophrenia diagnosis or of reporting symptoms of psychosis. These relations persisted after controlling for confounding variables, such as personal characteristics and other drug use. The relation did not seem to be a result of cannabis use to self-medicate symptoms of psychosis. A contributory causal relation is biologically plausible because psychotic disorders involve disturbances in the dopamine neurotransmitter systems with which the cannabinoid system interacts, as demonstrated by animal studies and one human provocation study. Conclusion: It is most plausible that cannabis use precipitates schizophrenia in individuals who are vulnerable because of a personal or family history of schizophrenia.

Mitochondrial dysmorphology in the neuroepithelium of rat embryos following a single dose of maternal hyperthermia during gestation

Hyperthermia is teratogenic to human and animal embryos and induces mainly anomalies of the nervous system. However, the teratogenic mechanism is poorly understood. Mammalian embryos are known to switch from anaerobic to aerobic metabolism around the time of neural tube closure. This critical event might be sensitive to hyperthermia. The objective of the present study was to evaluate the ultrastructural changes of the mitochondria of the neuroepithelium (NE) of rat embryos following maternal exposure to hyperthermia. Pregnant rats were heat stressed for an hour on gestation day (GD) 9 and embryos were examined by electron microscopy on GD 10. NE presented extensive apoptosis. Intercellular junctions were weakened and copious cellular debris projected into the ventricle. The mitochondria were of diverse size and shape. Most of them were swollen and had short cristae and electron dense matrix. Hydropic changes were also observed in numerous mitochondria. Lipid-laden mitochondria were found in the apical portions of neuroblasts. The mesenchyme (ME) of heat-treated embryos showed paucity of cells and only as frequent apoptosis as the controls. Their mitochondria also showed changes similar to those of the NE. Additionally extensive lipid accumulation was observed in and in the vicinity of mitochondria, often surrounded by short strands of endoplasmic reticulum. Whereas mitochondrial pathology was associated with profound apoptosis in the NE, growth restriction and lipid accumulation accompanied mitochondrial changes in the ME. The results of this study indicate that the embryonic response to maternal heat shock is tissue-specific and morphologically distinct in this species.

Protein kinase C activation and its role in kidney disease

Protein kinase C (PKC) comprises a superfamily of isoenzymes, many of which are activated by cofactors such as diacylglycerol and phosphatidylserine. In order to be capable of activation, PKC must first undergo a series of phosphorylations. In turn, activated PKC phosphorylates a wide variety of intracellular target proteins and has multiple functions in signal transduced cellular regulation. A role for PKC activation had been noted in several renal diseases, but two that have had most investigation are diabetic nephropathy and kidney cancer. In diabetic nephropathy, an elevation in diacylglycerol and/or other cofactor stimulants leads to an increase in activity of certain PKC isoforms, changes that are linked to the development of dysfunctional vasculature. The ability of isoform-specific PKC inhibitors to antagonize diabetes-induced vascular disease is a new avenue for treatment of this disorder. In the development and progressive invasiveness of kidney cancer, increased activity of several specific isoforms of PKC has been noted. It is thought that this may promote the kidney cancer's inherent resistance to apoptosis, in natural regression or after treatments, or it may promote the invasiveness of renal cancers via cellular differentiation pathways. In general, however, a more complete understanding of the functions of individual PKC isoforms in the kidney, and development or recognition of specific inhibitors or promoters of their activation, will be necessary to apply this knowledge for treatment of cellular dysregulation in renal disease.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the 131 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Intrauterine growth restriction due to uteroplacental vascular insufficiency leads to increased hypoxia-induced cerebral apoptosis in newborn piglets

Uteroplacental vascular insufficiency in humans is a common cause of intrauterine growth restriction (IUGR) and is associated with an increased incidence of perinatal asphyxia and neurodevelopmental disorders compared to normal weight newborns. Experimental models that provide an opportunity to analyze the pathogenesis of these relationships are limited. Here, we used neonatal pigs from large litters in which there were piglets of normal birth weight (for controls) and of low birth weight (for uteroplacental vascular insufficiency). Hypoxia was induced in paired littermates by reducing the fraction of inspired oxygen to 4% for 25 min. Brain tissue was collected 4 h post-hypoxia. Cerebral levels of apoptosis were quantified morphologically and verified with caspase-3 activity and TUNEL. Expression of Bcl-2, BcI-XL and Bax proteins was investigated using immunohistochemistry. Cellular positivity for Bcl-2 was consistently higher in the non-apoptotic white matter of the hypoxic IUGR animals compared with their littermates and reached significance at P < 0.05 in several pairs of littermates. Alterations in Bax showed a trend towards higher expression in the hypoxic IUGR littermates but rarely reached significance. The IUGR piglets showed a significantly greater amount of apoptosis in response to the hypoxia than the normal weight piglets, suggesting an increased vulnerability to apoptosis in the IUGR piglets. (c) 2006 Elsevier B.V. All rights reserved.

Increased apoptosis of T lymphocytes and macrophages in the central and peripheral nervous systems of Lewis rats with experimental autoimmune encephalomyelitis treated with dexamethasone

Using light and electron microscopic histological and immunocytochemical techniques, we investigated the effects of the glucocorticoid dexamethasone on T cell and macrophage apoptosis in the central nervous system (CNS) and peripheral nervous system (PNS) of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP). A single subcutaneous injection of dexamethasone markedly augmented T cell and macrophage apoptosis in the CNS and PNS and microglial apoptosis in the CNS within 6 hours (h). Pre-embedding immunolabeling revealed that dexamethasone increased the number of apoptotic CD5+ cells (T cells or activated B cells), αβ T cells, and CD11b+ cells (macrophages/microglia) in the meninges, perivascular spaces, and CNS parenchyma. The induction of increased apoptosis was dose-dependent. Daily dexamethasone treatment suppressed the neurological signs of EAE. However, the daily injection of a dose of dexamethasone (0.25 mg/kg). which, after a single dose, did not induce increased apoptosis in the CNS or PNS, was as effective in inhibiting the neurological signs of EAE as the high dose (4 mg/kg), which induced a marked increase in apoptosis. This indicates that the beneficial clinical effect of glucocorticoid therapy in EAE does not depend on the induction of increased apoptosis. The daily administration of dexamethasone for 5 days induced a relapse that commenced 5 days after cessation of treatment, with the severity of the relapse tending to increase with dexamethasone dosage.

p53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen-induced hepatotoxicity

Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cause of liver failure in humans. The NaS1 sulfate transporter maintains blood sulfate levels sufficiently high for sulforiation reactions to work effectively for drug detoxification. In the present study, we identified two loss-of-function polymorphisms in the human NaS1 gene and showed the Nas1-null mouse to be hypersensitive to APAP hepatotoxicity. APAP treatment led to increased liver damage and decreased hepatic glutathione levels in the hyposulfatemic Nas1-null mice compared with that in normosulfatemic wild-type mice. Analysis of urinary APAP metabolites revealed a significantly lower ratio of APAP-sulfate to APAP-glucuronide in the Nas1-null mice. These results suggest hyposulfatemia increases sensitivity to APAP-induced hepatotoxicity by decreasing the sulfonation capacity to metabolize APAP. In conclusion, the results of this study highlight the importance of plasma sulfate level as a key modulator of acetaminophen metabolism and suggest that individuals with reduced NaS1 sulfate transporter function would be more sensitive to hepatotoxic agents.

Drug targets and mechanisms of resistance in the anaerobic protozoa

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated ruing laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Cationic drug pharmacokinetics in diseased livers determined by fibrosis index, hepatic protein content, microsomal activity, and nature of drug

The disposition kinetics of six cationic drugs in perfused diseased and normal rat livers were determined by multiple indicator dilution and related to the drug physicochemical properties and liver histopathology. A carbon tetrachloride (CCl4)induced acute hepatocellular injury model had a higher fibrosis index (FI), determined by computer-assisted image analysis, than did an alcohol-induced chronic hepatocellular injury model. The alcohol-treated group had the highest hepatic alpha(1)- acid glycoprotein, microsomal protein (MP), and cytochrome P450 (P450) concentrations. Various pharmacokinetic parameters could be related to the octanol-water partition coefficient (log P-app) of the drug as a surrogate for plasma membrane partition coefficient and affinity for MP or P450, the dependence being lower in the CCl4-treated group and higher in the alcohol-treated group relative to controls. Stepwise regression analysis showed that hepatic extraction ratio, permeability-surface area product, tissue-binding constant, intrinsic clearance, partition ratio of influx (k(in)) and efflux rate constant (k(out)), and k(in)/k(out) were related to physicochemical properties of drug (log P-app or pK(a)) and liver histopathology (FI, MP, or P450). In addition, hepatocyte organelle ion trapping of cationic drugs was evident in all groups. It is concluded that fibrosis-inducing hepatic disease effects on cationic drug disposition in the liver may be predicted from drug properties and liver histopathology.

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved, effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.