Synthesis and biological evaluation of 3-(4-chlorophenyl)-4-substituted pyrazole derivatives.

3-(4-Chlorophenyl)-4-substituted pyrazole derivatives were synthesised and tested for their in vitro antifungal activity. Some compounds showed very good antifungal activity against four pathogenic strains of fungi. The same compounds exhibited an interesting activity against the tested strain of Mycobacterium tuberculosis H37Rv. The results suggest that 1,3,4-oxadiazoles and 5-pyrazolinones bearing a core pyrazole scaffold may be promising antifungal and antitubercular agents.

Long non-coding RNA GAS5 regulates apoptosis in prostate cancer cell lines.

While the role of small non-coding RNAs, such as miRNAs, in apoptosis control is well established, long non-coding RNAs (lncRNAs) have received less attention. Growth Arrest-Specific 5 (GAS5) encodes multiple snoRNAs within its introns, while exonic sequences produce lncRNA which can act as a riborepressor of the glucocorticoid and related receptors. GAS5 negatively regulates the survival of lymphoid and breast cells, and is aberrantly expressed in several cancers. Although cellular GAS5 levels decline as prostate cancer cells acquire castration-resistance, the influence of GAS5 on prostate cell survival has not been determined. To address this question, prostate cell lines were transfected with GAS5-encoding plasmids or GAS5 siRNAs, and cell survival was assessed. Basal apoptosis increased, and cell survival decreased, after transfection of 22Rv1 cells with plasmids encoding GAS5 transcripts, including mature GAS5 lncRNA. Similar effects were observed in PC-3 cells. In stable clones of 22Rv1, cell death correlated strongly with cellular GAS5 levels. Induction of 22Rv1 cell death by UV-C irradiation and chemotherapeutic drugs was augmented in cells transiently transfected with GAS5 constructs, and attenuated following down-regulation of GAS5 expression. Again, in these experiments, cell death was strongly correlated with cellular GAS5 levels. Thus, GAS5 promotes the apoptosis of prostate cells, and exonic sequence, i.e. GAS5 lncRNA, is sufficient to mediate this activity. Abnormally low levels of GAS5 expression may therefore reduce the effectiveness of chemotherapeutic agents. Although several lncRNAs have recently been shown to control cell survival, this is the first report of a death-promoting lncRNA in prostate cells.

GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer.

Effective control of both cell survival and cell proliferation is critical to the prevention of oncogenesis and to successful cancer therapy. Using functional expression cloning, we have identified GAS5 (growth arrest-specific transcript 5) as critical to the control of mammalian apoptosis and cell population growth. GAS5 transcripts are subject to complex post-transcriptional processing and some, but not all, GAS5 transcripts sensitize mammalian cells to apoptosis inducers. We have found that, in some cell lines, GAS5 expression induces growth arrest and apoptosis independently of other stimuli. GAS5 transcript levels were significantly reduced in breast cancer samples relative to adjacent unaffected normal breast epithelial tissues. The GAS5 gene has no significant protein-coding potential but expression encodes small nucleolar RNAs (snoRNAs) in its introns. Taken together with the recent demonstration of tumor suppressor characteristics in the related snoRNA U50, our observations suggest that such snoRNAs form a novel family of genes controlling oncogenesis and sensitivity to therapy in cancer.

Follow up of patients with colorectal polyps: are the BSG guidelines being adhered to?

OBJECTIVE: The publication of the BSG guidelines in 2002 provided a framework for the follow up of patients with colorectal polyps. The aim of the present study was to determine whether they had, or were being correctly adhered to in a moderately sized District General Hospital. METHOD: A total of 598 patients were on the waiting list for colonoscopy at Airedale General Hospital (AGH) in February 2005. Of these, 203 were being followed up as a result of the previous finding of a polyp. RESULTS: Only 14.8% of patients had been or were being followed up according to the BSG guidelines. The majority of the 85.2% of patients who did not comply with follow up did so as a result of over investigation. Seventy-eight per cent of the low-risk group and 55% of the intermediate-risk group had been colonoscoped, or were waiting to have colonoscopy, too soon or too frequently according to the BSG guidelines. Twenty-four patients with hyperplastic polyps were being followed up incorrectly, as were 17 patients discovered to have a polyp pathology on flexible sigmoidoscopy. It was established that 131 extra colonoscopies had been, or were planned to be performed unnecessarily. CONCLUSION: These data have major implications with regard to patient safety, service provision and cost to the NHS.

Psychiatric symptoms and problem behaviours in people with intellectual disabilities.

BACKGROUND: Previous studies have suggested different patterns of associations between psychiatric symptoms and problem behaviours in people with intellectual disabilities (ID). The aim of this study was to investigate which problem behaviours are associated with specific psychiatric symptoms and the relative strength of these specific associations. METHOD: A cross sectional survey using the Psychiatric Assessment Schedule for Adults with Developmental Disabilities Checklist and the Disability Assessment Schedule was carried out in a sample of 214 adults with ID. RESULTS: Self-injurious and, to a lesser extent, aggressive problem behaviours were most associated with affective type symptoms. Screaming and destructive behaviours tended to be more associated with autism-related social impairment rather than conventional psychiatric symptoms. CONCLUSIONS: This study gives further evidence of associations between psychiatric symptoms and specific problem behaviours in people with ID. It may be particularly useful to consider the diagnosis of affective disorders if a person with ID shows self-injurious or aggressive behaviours.

Maternal hypothyroidism in the rat influences placental and liver glycogen stores: fetal growth retardation near term is unrelated to maternal and placental glucose metabolic compromise.

Maternal hypothyroidism impairs fetal growth in the rat, but the mechanisms by which this occurs are unknown. Since the fetus derives its glucose supply from the mother, and maternal thyroidectomy may disturb maternal and placental glucose metabolism, we postulated that maternal and/or placental glucose metabolic compromise may contribute to fetal growth retardation in hypothyroid dams. Feto-placental growth, tissue glycogen stores and glucose levels in sera and amniotic fluid were determined in rat dams partially thyroidectomized (TX) before pregnancy and in euthyroid controls. Fetal body weight at 16, 19 and 21 days gestation (d.g.) was related to pre-mating maternal serum total thyroxine (TT(4)) levels; permanent fetal growth retardation occurred in severely (TX(s); pre-mating maternal serum TT(4)

Influence of maternal hyperthyroidism in the rat on the expression of neuronal and astrocytic cytoskeletal proteins in fetal brain.

Maternal hypothyroidism during pregnancy impairs brain function in human and rat offspring, but little is known regarding the influence of maternal hyperthyroidism on neurodevelopment. We have previously shown that the expression of neuronal and glial differentiation markers in fetal brain is compromised in hypothyroid rat dam pregnancies and have now therefore extended this investigation to hyperthyroid rat dams. Study groups comprised partially thyroidectomised dams, implanted with osmotic pumps infusing either vehicle (TX dams) or a supraphysiological dose of thyroxine (T4) (HYPER dams), and euthyroid dams infused with vehicle (N dams). Cytoskeletal protein abundance was determined in fetal brain at 21 days of gestation by immunoblot analysis. Relative to N dams, circulating total T4 levels were reduced to around one-third in TX dams but were doubled in HYPER dams. Fetal brain weight was increased in HYPER dams, whereas litter size and fetal body weight were reduced in TX dams. Glial fibrillary acidic protein expression was similar in HYPER and TX dams, being reduced in both cases relative to N dams. alpha-Internexin (INX) abundance was reduced in HYPER dams and increased in TX dams, whereas neurofilament 68 (NF68) exhibited increased abundance in HYPER dams. Furthermore, INX was inversely related to - and NF68 directly related to - maternal serum total T4 levels, independently of fetal brain weight. In conclusion, maternal hyperthyroidism compromises the expression of neuronal cytoskeletal proteins in late fetal brain, suggestive of a pattern of accelerated neuronal differentiation.

Enhanced activity by poly(ethylene glycol) modification of Coriolopsis gallica laccase.

We are studying the enzymatic modification of polycyclic aromatic hydrocarbons (PAHs) by the laccase from Coriolopsis gallica UAMH 8260. The enzyme was produced during growth in a stirred tank reactor to 15 units ml(-1), among the highest levels described for a wild-type fungus; the enzyme was the major protein produced under these conditions. After purification, it exhibited characteristics typical of a white rot fungal laccase. Fifteen azo and phenolic compounds at 1 mM concentration were tested as mediators in the laccase oxidation of anthracene. Higher anthracene oxidation was obtained with the mediator combination of ABTS and HBT, showing a correlation between the oxidation rate and the mediator concentration. Reactions with substituted phenols and anilines, conventional laccase substrates, and PAHs were compared using the native laccase and enzyme preparations chemically modified with 5000 MW-poly(ethylene glycol). Chemically modified laccase oxidized a similar range of substituted phenols as the native enzyme but with a higher catalytic efficiency. The k(cat) increase by the chemical modification may be as great as 1300 times for syringaldazine oxidation. No effect was found of chemical modification on mediated PAH oxidation. Both unmodified and PEG-modified laccases increased PAH oxidation up to 1000 times in the presence of radical mediators. Thus, a change of the protein surface improves the mediator oxidation efficiency, but does not affect non-enzymatic PAH oxidation by oxidized mediators.

Effect of growth conditions on the production of manganese peroxidase by three strains of Bjerkandera adusta.

We were looking for a strain of Bjerkandera adusta that produces high titres of manganese peroxidase under optimal conditions for large-scale enzyme purification. We have chosen two strains from the University of Alberta Microfungus Collection and Herbarium, UAMH 7308 and 8258, and compared the effects of growth conditions and medium composition on enzyme production with the well-characterized strain BOS55 (ATCC 90940). Of four types of cereal bran examined, rice bran at 3% (w/v) in 60 mM phosphate buffer pH 6 supported the highest levels of enzyme production. Using 100 mL medium in 500-mL Erlenmeyer flasks, maximum enzyme levels in the culture supernatant occurred after about 10 days of growth; 5.5 U x mL(-1) for UAMH 7308, 4.4 U x mL(-1) for UAMH 8258, and 1.7 U x mL(-1) for BOS55, where units are expressed as micromoles of Mn-malonate formed per minute. Growth as submerged cultures in 10-L stirred tank reactors produced 3.5 U x mL(-1) of manganese peroxidase (MnP) by UAMH 8258 and 2.5 U x mL(-1) of MnP by 7308, while enzyme production by BOS55 was not successful in stirred tank reactors but could be scaled up in 2-L shake flasks containing 400 mL rice bran or glucose-malt-yeast extract (GMY)-Mn-glycolate medium to produce MnP levels of 1.7 U x mL(-1). These results show that the two strains of B. adusta, UAMH 7308 and 8258, can produce between two and three times the manganese peroxidase level of B. adusta BOS55, that they are good candidates for scale up of enzyme production, and that the rice bran medium supports higher levels of enzyme production than most previously described media.

Kinetic differences of purified laccases from six Pleurotus ostreatus strains.

AIMS: Enzyme kinetics of purified laccases from six different Pleurotus ostreatus strains were determined in the oxidation of syringaldazine, guaiacol and ABTS. METHODS AND RESULTS: Significant differences in the kinetic constants were found. Catalytic activity (kcat) ranged from 19 to 941 U mg(-1) for syringaldazine, from 18 to 1565 U mg(-1) for ABTS, and from 4 to 44 U mg(-1) for guaiacol. The apparent affinity constants (KM) also showed significant differences between the different strains, from 12 to 52 micromol l(-1) for syringaldazine, from 8 to 79 micromol l(-1) for ABTS, and from 0.46 to 6.61 mmol l(-1) for guaiacol. No differences were found either on the effect of increasing concentrations of organic solvent (acetonitrile) or on the activity pH profile. The temperature profile was the same for all the P. ostreatus strains, except for the IE8 strain, which seems to be more sensitive to temperature. The kinetic and stability data from the six P. ostreatus strains were also compared with those obtained from other white rot fungi, Coriolopsis gallica and Trametes versicolor, showing clear differences. CONCLUSION: The different P. ostreatus isolates showed different kinetic constants. SIGNIFICANCE AND IMPACT OF THE STUDY: The different enzymatic properties of laccases from various P. ostreatus strains should be considered for a potential industrial or environmental application.

Thyroid hormone receptor expression in rat placenta.

The expression of c- erbAalpha and -beta encoded thyroid hormone receptors (TR) was investigated in rat placenta between 16 and 21 days of gestation (dg), and in fetal liver and brain at 16 dg, using semi-quantitative RT-PCR and nuclear 3,5,3'-triiodothyronine (T(3)) binding. TRalpha1, TRbeta1, c- erbAalpha 2 and c- erbAalpha 3 mRNA abundance was unchanged in placenta between 16 and 21 dg, as was the dissociation constant (K(d)) of T(3) binding. The maximal T(3) binding capacity (B(max)) in placenta doubled over this period, suggesting placental TR binding activity is post-transcriptionally regulated. Transcript abundance in tissues at 16 dg can be summarized: TRalpha1, placenta=fetal liverfetal brain; c- erbAalpha 2 and alpha3, placenta=fetal liver

Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a.

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.

Maternal thyroid status regulates the expression of neuronal and astrocytic cytoskeletal proteins in the fetal brain.

Maternal thyroid hormone (TH) crosses the placenta and is postulated to regulate fetal brain development. However, TH-dependent stages of fetal brain development remain to be characterised. We have therefore compared the levels of several neuronal and glial cytoskeletal proteins in fetal brains from normal (N) and partially thyroidectomised (TX) rat dams by immunoblotting. Pregnancies were studied both before and after the onset of fetal TH secretion, which occurs at 17.5 days gestation (dg) in the rat. Maternal hypothyroidism disrupted fetal growth, so that fetal body and brain weights were reduced near term. Vimentin expression was unaffected, however, indicating normal acquisition of neuronal and glial precursor cells. Fetal brain levels of glial fibrillary acidic protein (GFAP) were reduced at 21 dg, suggesting delayed astrocytic differentiation, although regression analysis demonstrated appropriate GFAP levels for brain weight. Levels of alpha-internexin, the earliest neurofilament protein expressed in fetal brain were reduced at 16 dg in TX dams, but increased at 21 dg. The ontogeny of neurofilament-L was also perturbed in these pregnancies, with deficient levels apparent at both 16 and 21 dg. These effects on neuronal cytoskeletal proteins were unrelated to fetal brain growth retardation. These findings confirm that maternal hypothyroidism disrupts early fetal brain development. Early disturbances in neuronal differentiation are not corrected by the onset of fetal TH secretion. Such disturbances may contribute to the neurological damage observed in children born to hypothyroxinaemic mothers.

Thyroid hormone receptor gene expression in first trimester human fetal brain.

Maternal thyroid hormone is transferred to the fetus early in pregnancy and is postulated to regulate brain development. Thyroid hormone nuclear receptor (TR) proteins are present in fetal brain, but their isoformal composition is unknown. We therefore investigated the ontogeny of TR isoforms and related splice variants in first trimester human fetal brain (n = 9) by semi-quantitative reverse transcriptase-polymerase chain reaction analysis. Expression of the TRbeta1, TRalpha1 and c-erbAalpha2 isoforms was detected from 8.1 weeks gestation (wg). An additional truncated species was detected with the c-erbAalpha2 primer set, consistent with the c-erbAalpha3 splice variant previously described in the rat. All c-erbAalpha-derived transcripts were co-ordinately expressed and increased (ca. 8-fold) between 8.1 and 13.9 wg. A more complex ontogenic pattern was observed for TRbeta1, suggestive of a nadir between 8.4 and 12.0 wg. These findings point to an important role for the TRalpha1 isoform in mediating maternal thyroid hormone action during first trimester human fetal brain development.

Low surfactant concentration increases fungal mineralization of a polychlorinated biphenyl congener but has no effect on overall metabolism.

Three white rot fungi were compared for their ability to attack polychlorinated biphenyl (PCB) congeners in the presence and absence of the non-ionic Triton X-100 or the anionic Dowfax 8390 surfactants at half their critical micelle concentrations. Neither surfactant affected PCB biodegradation monitored by gas chromatography but the release of 14CO2 from 2,4',5-[U-14C]trichlorobiphenyl by Trametes versicolor was stimulated 12% by Triton X-100. Since mineralization is the complete metabolism of the congener and biodegradation was measured as substrate disappearance, Triton X-100 is proposed to aid intracellular solubilization of 2,4',5-trichlorobiphenyl for complete oxidation by T. versicolor.

Alcohol and drug use in second-year medical students at the University of Leeds.

OBJECTIVE: In view of recent media attention concerning the high level of alcohol and drug use reported in a group of newly qualified junior doctors, the aim of the present study was to assess the alcohol and drug habits of a group of current medical students. METHODS: Information about alcohol and illicit drug use was obtained from 136 second-year medical students (46 men, 90 women) at the University of Leeds by means of a personally administered questionnaire. Levels of anxiety and depression were also assessed. RESULTS: 86% of the students drank alcohol and among those who drank, a high proportion (52.6% of the men and 50.6% of the women), exceeded the recommended weekly limit of alcohol consumption of 21 units for men and 14 units women per week). Illicit drug use was reported by 33.1% of students (28.3% of men, 35.6% of women). The drug most commonly used was cannabis. According to the Hospital Anxiety and Depression (HAD) scale, 41.2% of the students (37% of men, 43.3% of women) had anxiety ratings within the clinically significant range (HAD scale> 8); 9.5% of students demonstrated clinically significant levels of depression (HAD scale> 8). However, these high levels of anxiety and depression did not correlate with high levels of alcohol consumption or drug use.

Surfactant inhibition of bacterial growth on solid anthracene.

Surfactants have been proposed as a promising method to enhance bioremediation of hydrophobic compounds in contaminated soils. However, the results of effects of surfactants on bioremediation are not consistent. This study showed that Triton X-100 at low concentration (0.024 mM or 0.09 CMC) inhibited the rate of growth of either a Mycobacterium sp. or a Pseudomonas sp. on solid anthracene as sole carbon source. Recovery of microbial growth rate could be achieved by dilution of surfactants, while addition of more surfactant gave an immediate decrease in growth rate. No inhibition of growth by Triton X-100 was observed with growth on glucose. The surfactant sorbed onto the surfaces of both the cells and the anthracene particles, which could inhibit uptake of anthracene. The results were consistent with the hypothesis that inhibition of microbial adhesion of cells to anthracene was responsible for the inhibition of growth by Triton X-100.

Maternal hypothyroxinemia influences glucose transporter expression in fetal brain and placenta.

The influence of maternal hypothyroxinemia on the expression of the glucose transporters, GLUT1 and GLUT3, in rat fetal brain and placenta was investigated. Fetal growth was retarded in hypothyroxinemic pregnancies, but only before the onset of fetal thyroid hormone synthesis. Placental weights were normal, but placental total protein concentration was reduced at 19 days gestation (dg). Immunoblotting revealed a decreased abundance of GLUT1 in placental microsomes at 16 dg, whereas GLUT3 was increased. Fetal serum glucose levels were reduced at 16 dg. In fetal brain, the concentration of microsomal protein was deficient at 16 dg and the abundance of parenchymal forms of GLUT1 was further depressed, whereas GLUT3 was unaffected. Northern hybridization analysis demonstrated normal GLUT1 mRNA levels in placenta and fetal brain. In conclusion, maternal hypothyroxinemia results in fetal growth retardation and impaired brain development before the onset of fetal thyroid function. Glucose uptake in fetal brain parenchyma may be compromised directly, due to deficient GLUT1 expression in this tissue, and indirectly, as a result of reduced placental GLUT1 expression. Though corrected by the onset of fetal thyroid hormone synthesis, these deficits are present during the critical period of neuroblast proliferation and may contribute to long term changes in brain development and function seen in this model and in the progeny of hypothyroxinemic women.

SHORT COMMUNICATION maternal thyroid dysfunction and c- fos and c- jun expression in rat placenta.

Maternal thyroid dysfunction is associated with perturbed fetal brain development and neurological deficits in adulthood in rat and human. To investigate whether these effects occur secondary to placental dysfunction, c- fos and c- jun expression in placenta from normal (euthyroid) and moderately hypothyroid rat dams were investigated by Northern hybridization analysis. In normal placenta, c- fos expression increased by 74 per cent between 16 and 21 days of gestation (dg) whereas c- jun expression declined by 46 per cent. Moderate maternal hypothyroidism depressed placental c- fos expression by 32 per cent at 19 dg, but elevated c- fos and c- jun expression by 139 and 86 per cent, respectively, at 21 dg. Maternal hypothyroidism may therefore induce c- fos/c- jun -related placental dysfunction, but only relatively late in gestation when fetal thyroid function is already established.

Polycyclic aromatic hydrocarbon metabolism by white rot fungi and oxidation by Coriolopsis gallica UAMH 8260 laccase.

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 microg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from Coriolopsis gallica. The reaction mixtures contained 20 microM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene. There was no clear relationship between the ionization potential of the substrate and the first-order rate constant (k) for substrate loss in vitro in the presence of ABTS. The effects of mediating substrates were examined further by using anthracene as the substrate. Hydroxybenzotriazole (HBT) (1 mM) supported approximately one-half the anthracene oxidation rate (k = 2.4 h(-1)) that ABTS (1 mM) supported (k = 5.2 h(-1)), but 1 mM HBT plus 1 mM ABTS increased the oxidation rate ninefold compared with the oxidation rate in the presence of ABTS, to 45 h(-1). Laccase purified from Pleurotus ostreatus had an activity similar to that of C. gallica laccase with HBT alone, with ABTS alone, and with 1 mM HBT plus 1 mM ABTS. Mass spectra of products obtained from oxidation of anthracene and acenaphthene revealed that the dione derivatives of these compounds were present.