Specificity of 17 beta-oestradiol and benzo[a] pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg(48)Ala(119)Leu(432), Arg(48)Ala(119)Val(432), Gly(48)Ala(119)Leu(432), Gly(48)Ala(119)Val(432), Arg(48)Ser(119)Leu(432), Arg(48)Ser(119)Val(432), Gly(48)Ser(119)Leu(432) and Gly(48)Ser(119)Val(432) (all with Asn(453)), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17 beta -oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+. CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg(48) variants tended to have higher activities for 17 beta -oestradiol 4-hydroxylation than Gly(48) variants, although there were no significant variations in 17 beta -oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17 beta -oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val(432) forms than the corresponding Leu(432) forms. 4. In contrast, Leu(432) forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7, 8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val(432) forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17 beta -oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Long-run growth effects of taxation in a non-scale growth model with innovation

In previous studies, taxing income or consumption hinders long-run growth. Incorporating saving and leisure into the non-scale Schumpeterian model of [Journal of Political Economy 107 (1999) 715-730], we show that the usual growth effects of taxing consumption and labor income do not exist. (C) 2002 Elsevier Science B.V. All rights reserved.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Human immunodeficiency virus type 1 reverse transcription is stimulated by Tat from other lentiviruses

The tat gene is required by HIV-1 for efficient reverse transcription and this function of Tat can be distinguished from its role in transcription by RNA polymerase II using tat point mutations that abrogate each function independently The mechanism of Tat's role in reverse transcription, however, is not known, nor is it known whether this role is conserved among trans-activating factors in other retroviruses. Here we examine the abilities of heterologous viral trans-activating proteins from jembrana disease virus (jTat), HIV-2 (Tat2), and equine infectious anemia virus (eTat) to substitute for HIV-1 Tat (Tat1) and restore reverse transcription in HIV-1 carrying an inactivated tat gene. Natural endogenous reverse transcription assays showed that trans-activators from some retroviruses (Tat2 and jTat, but not eTat) could substitute for Tat1 in complementation of HIV-1 reverse transcription. Finally, we show that Y47 is critical for Tat1 to function in reverse transcription, but not HIV-1 gene expression. We mutated the homologous position in jTat to H62Y and found it did not improve its ability to stimulate reverse transcription, but an H62A mutation did inhibit jTat complementation. These data highlight the finding that the role of Tat in reverse transcription is not related to trans-activation and demonstrate that other tat genes conserve this function. (C) 2002 Elsevier Science (USA).

Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.

Inhibition of adventitious rooting in Backhousia citriodora F. Muell cuttings correlate with the concentration of essential oil

Backhousia citriodora is typical of the many commercially valuable woody Australian Myrtaceae species that are recalcitrant in forming adventitious roots from cuttings after maturation. A series of experiments were conducted to identify an endogenous rooting inhibitor in line with established criteria. Endogenous levels of citral were correlated with the rooting capacities of juvenile versus mature, and easy- versus difficult-to-root genotypes of B. citriodora, in both winter and summer. The biological activity of citral was confirmed in bioassays on mung beans and easy-to-root B. citriodora seedlings. Evidence of a common mechanism of root inhibition with other species in the Myrtaceae and the role of action of citral are discussed.

Oral administration of a 12% sucrose solution did not decrease behavioural indicators of distress in piglets undergoing tail docking, teeth clipping and ear notching

Sucrose has been shown to attenuate the behavioural response to painful procedures in human infants undergoing circumcision or blood collection via heelstick. Sucrose has also been found to have a behaviour-modifying effect in neonatal rats exposed to a hot plate. The effect was abolished in neonatal rats by injection of the opioid antagonist naltrexone, suggesting that it was mediated by endogenous opioids. In this experiment, the behaviour of 571 newborn Large White x Landrace hybrid piglets in a specific-pathogen-free piggery of the University of Queensland was recorded during and after the routine management practices of tail docking, ear notching and teeth clipping. Piglets were randomly assigned to receive 1.0 ml of a 12% sucrose solution (treatment group) or a placebo (1.0 ml of air) administered via syringe in the mouth, 60 s before commencement of one of the management procedures. Behaviours were recorded at the time of the procedure, and then 2 min after completion of the procedure. Piglets that received the sucrose solution did not behave significantly differently from piglets receiving the placebo. Regardless of whether sucrose or placebo was administered, piglets undergoing the routine management procedures showed significantly greater behavioural responses than piglets undergoing no procedure. It was concluded that under commercial conditions, a 12% sucrose solution administered I min prior to surgery was not effective in decreasing the behavioural indicators of distress in piglets undergoing routine management procedures, Further research into methods of minimising distress caused to piglets by these procedures is recommended.

The coactivator-associated arginine methyltransferase is necessary for muscle differentiation - CARM1 coactivates myocyte enhancer factor-2

Studies with the myogenic basic helix-loop-helix and MADS box factors suggest that efficient transactivation is dependent on the recruitment of the steroid receptor coactivator (SRC) and the cofactors p300 and p300/CBP-associated factor. SRCs have been demonstrated to recruit CARM1 (coactivator-associated arginine methyltransferase-1), a member of the S-adenOSyl-L-methionine-dependent PRMTI-5 (protein-arginine N-methyltransferase-1-5) family, which catalyzes the methylation of arginine residues. This prompted us to investigate the functional role of CARM1/PRMT4 during skeletal myogenesis. We demonstrate that CARM1 and the SRC cofactor GRIP-1 cooperatively stimulate the activity of myocyte enhancer factor-2C (MEF2C). Moreover, there are direct interactions among MEF2C, GRIP-1, and CARM1. Chromatin immunoprecipitation demonstrated the in vivo recruitment of MEF2 and CARM1 to the endogenous muscle creatine kinase promoter in a differentiation-dependent manner. Furthermore, CARM1 is expressed in somites during embryogenesis and in the nuclei of muscle cells. Treatment of myogenic cells with the methylation inhibitor adenosine dialdehyde or tet-regulated CARM1 antisense expression did not affect expression of MyoD. However, inhibition of CARM1. inhibited differentiation and abrogated the expression of the key transcription factors (myogenin and MEF2) that initiate the differentiation cascade. This work clearly demonstrates that the arginine methyltransferase CARM1 potentiates myogenesis and supports the positive role of arginine methylation in mammalian differentiation.

Modification of induced ischaemic pain by placebo electrotherapy

Quantifying the analgesic effect of placebo electrotherapy is an important part of understanding the placebo response in physiotherapy. This repeated measures study of induced ischaemic pain compared reports of pain threshold, pain tolerance, and pain endurance under three conditions: control, placebo interferential, and placebo TENS. Both of the placebo conditions significantly delayed the report of pain threshold. Placebo interferential also delayed pain tolerance. Each placebo condition reduced pain intensity in the 6th minute. Only placebo TENS reduced pain at the 9th minute of ischaemic pain. The nature of pain reduction in the placebo conditions suggests that analgesia was due to learned expectancies and endogenous opioid release. Further research into the impact of positive expectancies of pain relief in our patients could clarify the efficacy of physiotherapy outcomes for pain.

Effect of varying levels of mental workload on startle eyeblink modulation

Previous research using punctuate reaction time and counting tasks has found that the startle eyeblink reflex is sensitive to attentional demands. The present experiment explored whether startle eyeblink is also modulated during a complex continuous task and is sensitive to different levels of mental workload. Participants (N=14) performed a visual horizontal tracking task either alone (single-task condition) or in combination with a visual gauge monitoring task (multiple-task condition) for three minutes. On some task trials, the startle eyeblink reflex was elicited by a noise burst. Results showed that startle eyeblink was attenuated during both tasks and that the attenuation was greater during the multiple-task condition than during the single-task condition. Subjective ratings, endogenous eyeblink rate, heart period, and heart period variability provided convergent validity of the workload manipulations. The findings suggest that the startle eyeblink is sensitive to the workload demands associated with a continuous visual task. The application of startle eyeblink modulation as a workload metric and the possibility that it may be diagnostic of workload demands in different stimulus modalities is discussed.

Peroxisome proliferator-activated receptor beta expression in human breast epithelial cell lines of tumorigenic and non-tumorigenic origin

Peroxisome proliferator-activated receptor beta (PPARbeta) is a member of the nuclear hormone receptor superfamily and is a ligand activated transcription factor. although the precise genes that it regulates and its physiological and pathophysiological role remain unclear. In view of the association of PPARbeta with colon cancer and increased mRNA levels of PPARbeta in colon tumours we sought in this study to examine the expression of PPARbeta in human breast epithelial cells of tumorigenic (MCF-7 and MDA-MB-231) and non-tumorigenic origin (MCF-10A). Using quantitative RT-PCR we measured PPARbeta mRNA levels in MCF-7. MDA-MB-231 and MCF-10A cells at various stages in culture. After serum-deprivation, MDA-MB-231 and MCF-10A cells had a 4.2- and 3.8-fold statistically greater expression of PPARbeta compared with MCF-7 cells. The tumorigenic cell lines also exhibited a significantly greater level of PPARbeta mRNA after serum deprivation compared with subconfluence whereas such an effect was not observed in non-tumorigenic MCF-10A cells. The expression of PPARbeta was inducible upon exposure to the PPARbeta ligand bezafibrate. Our results suggest that unlike colon cancer. PPARbeta overexpression is not an inherent property of breast cancer cell lines. However, the dynamic changes in PPARbeta mRNA expression and the ability of PPARbeta in the MCF-7 cells to respond to ligand indicates that PPARbeta may play a role in mammary gland carcinogenesis through activation of downstream genes via endogenous fatty acid ligands or exogenous agonists. (C) 2002 Elsevier Science Ltd. All rights reserved.

Patterns of lipid storage and mobilisation in the female green sea turtle (Chelonia mydas)

Reproductive data from southern Queensland indicate that vitellogenesis in female Chelonia mydas takes approximately 8 months and is followed by a migration to a breeding area. At Heron Island, females lay multiple clutches over approximately 3 months. To investigate how females mobilise and store lipid during the breeding season we collected plasma, yolk, and fat tissue samples from females at a variety of stages during the nesting season. In breeding females, concentrations of plasma triglyceride increased seasonally. They reached peak concentrations during vitellogenesis and courtship, remained high throughout the nesting season, and then declined to a nadir after the last clutch. Plasma protein concentration increased throughout the breeding season, peaking following the last clutch for the season. Yolk lipids were highest during courtship and were similar throughout the nesting season, suggesting that uptake of lipid by ovarian follicles is completed prior to the beginning of the nesting season. Plasma triglyceride decreases in females with prolonged periods of unsuccessful nesting, and total lipid levels in adipose tissue and follicle yolks were significantly lower in atretic females. It appears that: (1) endogenous energy reserves can be reduced by stochastic environmental events (such as those reducing nesting success), and (2) a metabolic shift signalling the end of the nesting season is characterised by a drop in plasma triglycerides and slight increase in total plasma protein.

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

A review of the flower characteristics of Geraldton waxflower and factors influencing their abscission from harvested stems

Geraldton waxflower (Chamelaucium uncinatum Schauer) is Australia's most economically important cut-flower export. Its small, attractive flowers make it particularly suitable as a filler in floral arrangements. However, postharvest bud and flower abscission is a major problem during transport, handling and marketing. Abscission may be caused by wound-induced endogenous ethylene production brought about by flower tissue infection with fungal pathogens such as Botrytis cinerea. Botany and postharvest characteristics are discussed in relation to flower abscission and how resultant postharvest losses may be minimised.

Conservation of the cardiostimulant effects of (-)-Norepinephrine across Ser49Gly and Gly389Arg Beta1-adrenergic receptor polymorphisms in human right atrium in vitro

OBJECTIVES The goal of this study was to determine whether the cardiostimulant effects of the endogenous beta(1)-adrenergic receptor (AR) agonist, (-)-norepinephrine are modified by polymorphic (Serine49Glycine [Ser49Gly], Glycine389Arginine [Gly389Arg]) variants of beta(1)-ARs in the nonfailing adult human heart. BACKGROUND Human heart beta(1)-ARs perform a crucial role in mediating the cardiostimulant effects of (-)-norepinephrine. An understanding of the significance of Ser49Gly and Gly389Arg polymorphisms in the human heart is beginning to emerge, but not as yet in adult patients who have coronary artery disease (CAD). METHODS The potency and maximal effects of (-)-norepinephrine at beta(1)-ARs (in the presence of beta(2)-AR blockade with 50 nM ICI 118,551 [erythro-DL-1(7-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol]) for changes in contractile force and shortening of contractile cycle duration were determined in human right atrium in vitro from 87 patients undergoing coronary artery bypass grafting who were taking beta-blockers before surgery. A smaller sample of patients (n = 20) not taking beta-blockers was also investigated. Genotyping for two beta(1)-AR polymorphisms (Ser49Gly and Gly389Arg) was determined from a sample of blood taken at the time of surgery. RESULTS (-)-Norepinephrine caused concentration-dependent increases in contractile force and reductions in time to reach peak force and time to reach 50% relaxation. There were no differences in the potency or maximal effects of (-)-norepinephrine in the right atrium from patients with different Ser49Gly and Gly389Arg polymorphisms. CONCLUSIONS The cardiostimulant effects of (-)-norepinephrine at beta(1)-ARs were conserved across Ser49Gly and Gly389Arg polymorphisms in the right atrium of nonfailing hearts from patients with CAD managed with or without beta-blockers. (C) 2002 by the American College of Cardiology Foundation.