Effect of ACL reconstruction tunnels on stress in the distal femur.

PURPOSE: This study examined the change in femoral stress caused by graft tunnels drilled for anterior cruciate ligament (ACL) reconstruction. Using a computational model, the number, geometry and position of the graft tunnels exits were varied to determine the effect on bone stress. METHODS: A finite element model of the distal femur was developed from a CT scan of a cadaveric knee. To assess the model, the strain calculated computationally was compared to experimentally measured strains in eleven unpaired human cadaver femurs. Using the computational model, the number, geometry and position of the graft tunnel exits were varied to determine the effect on bone stress based on the stress concentration factor: the ratio of bone stress with tunnels to intact bone stress. RESULTS: The results indicated that the second tunnel in double-bundle ACL reconstruction results in approximately a 20 % increase in the maximum femoral stress as compared to single-bundle reconstruction. The highest stresses occur at the tunnel exits. The position of the tunnel exits effects femoral stress with the stress increasing slightly (AM SCR from 0.7 to 1 and PL SCR from 1.2 to 1.3) when the AM tunnel exit is moved anteriorly and having greater increases as the posterior lateral (PL) tunnel exit is moved laterally (PL SCR from 1.2 to 1.7) or posteriorly (PL SCR from 1.2 to 2). CONCLUSION: In anatomical ACL reconstruction, the tunnel entrances are dictated by anatomy; however, there can be variations in tunnel exit positions. Consideration should be given when positioning tunnel exits on the effect on stress in the femur. Moving the PL tunnel exit laterally or posteriorly increases in the stress at the PL tunnel exit.

On-line determination and control of fat content in batches of beef trimmings by NIR imaging spectroscopy.

An NIR imaging scanner was calibrated for on-line determination of the fat content of beef trimmings. A good calibration model was obtained for fat in intact beef (R=0.98, RMSECV=3.0%). The developed model could be used on single pixels to get an image of the fat distribution, or on the average spectrum from each trimming/portion of trimmings passing under the scanner. The fat model gave a rather high prediction error (RMSEP=8.7%) and a correlation of 0.84 when applied to 45 single trimmings with average fat content ranging from 1.6 to 49.3% fat. Test measurements on streams of trimmings making up batches varying from 10 to 24 kg gave a much lower prediction error (RMSEP=1.33%). Simulations based on true measurements indicate that the RMSEP decreases with increasing batch size and, for the present case, reached about 0.6% for 100 kg batches. The NIR scanner was tested on six batches of intact trimmings varying from 145 to 210 kg and gave similar fat estimates as an established microwave system obtained on the ground batches. The proven concept should be applicable to on-line estimation of fat in trimmings in order to determine the batch fat content and also to control the production of batches to different target fat levels. A possible requirement for the concept to work properly is that the trimming or layer of trimmings on the belt is not too thick. In this study maximum thickness was about 8 cm. Thicker trimmings might be measured, but careful hardware adjustments are then required.

Evaluation of a community fissure sealant programme in County Meath, Ireland.

OBJECTIVE: To evaluate the fissure sealant programme operated by the North Eastern Health Board Dental Service in County Meath, Republic of Ireland. The fissure sealant programme forms part of the school dental service aimed at children in first class (age group 6-7 years). DESIGN: Cross sectional study with retrospective analysis of dental records. CLINICAL SETTING: Schools in County Meath in 1999. PARTICIPANTS: Children in fourth class (mean age 9.6 years) in the school year 1999/2000 who had participated in the fissure sealant programme in the school year 1996/97. MAIN OUTCOME MEASURES: Sealant retention using the criteria of Simonsen. RESULTS: The mean age of sealant was 2.3 years. Fifty six percent of sealants were completely retained, 27% were partially retained and 12.8% were missing. The majority (73%) of children had some sealant on at least one tooth. Caries experience in previously sealed teeth was low (2.9%). Children who had all four first permanent molars sealed had a significantly lower DMFT (visual) than those who had no sealants (Wilcoxan p < 0.0001). CONCLUSION: Despite lack of maintenance of sealants in this study, retention rates compared favourably with similar international studies and caries experience in previously sealed teeth was low. The use of a written sealant policy and protocol for sealant application and equipment maintenance could further improve retention rates. Children who had no sealants had significantly poorer dental health than children who had all four first permanent molars sealed.

Studies on the mechanism of enhancement of purinergic transmission by caffeine in the guinea-pig isolated vas deferens.

1. Purinergic transmission from sympathetic nerves in the guinea-pig vas deferens was monitored using intracellular recording techniques. Stimulation of the hypogastric nerve with trains of 15 pulses at 1 Hz evoked excitatory junction potentials (EJPs) which increased in amplitude from the first pulse and reached a maximum after 6-8 pulses. 2. Caffeine (3 and 10 mm), depolarized cells by 5-10 mV and increased the amplitude of the first few EJPs in each train but reduced the maximum amplitude of EJPs late in the train. 3. The adenosine receptor antagonist 8-p-sulphophenyl-theophylline (8-SPT; 30 microm) had no effect on either the resting membrane potential or the EJP amplitude; however, at 100 microm it reduced the amplitude of all EJPs by 5-10%. 4. Adenosine (10 and 30 microm) reduced the amplitude of EJPs in a concentration-dependent manner. The inhibitory effect of adenosine on EJP amplitude was prevented by pretreatment with either caffeine (3 mm) or 8-SPT (30 microm). 5. Ryanodine (30 microm) did not alter EJP amplitude and did not inhibit the enhancement of the first EJP by caffeine (3 mm). Incubation of the tissue with the cell permeable calcium chelator 1-2-bis(o-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid (BAPT-AM) resulted in a depression of EJP amplitude and a longer time to reach maximum amplitude. In cells that had been exposed to BAPT-AM, caffeine 3 mm still increased amplitude of EJP early in the train. 6. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 500 microm), hyperpolarized cells and increased the amplitude of EJP throughout the train of stimulation. In the presence of IBMX, caffeine 3 mm still depolarized the cells and enhanced the EJP early in the train of stimulation. 7. The findings in this study confirm that caffeine and 8-SPT are effective inhibitors of the actions of adenosine. However, caffeine has an additional action to enhance EJP early during a train of stimulation, which cannot be attributed to blockade of adenosine receptors, but which may be related to inhibition of phosphodiesterase.

Dental health and treatment needs among a sample of Bangladeshi medical users aged 40 years and over living in Tower Hamlets, UK.

AIM: To assess the dental health status and treatment needs of Bangladeshi medical care users aged 40 years and over and to explore the relationship of oral disease status, tobacco usage and paan (betel quid) chewing. DESIGN: Multi-centre cross-sectional study. SETTING: General medical practitioners' waiting areas in Tower Hamlets, UK. SUBJECTS: 185 Bangladeshi adults aged 40 years and over. INTERVENTION: A clinical examination and an interview schedule. MEASURES: Dental status, periodontal status, dental plaque, calculus and denture status. Tobacco smoking and paan chewing behaviour. RESULTS: The response rate was 74%. 85% of participants were dentate with an average number of 24 (SD +/- 5.4) standing teeth. The mean DMFT score was 5.38, with missing teeth (3.81) being the major component. The decayed component was 0.43 and the filled was 1.14. 46% of participants were assessed as being free from gingivitis. Significant relationships between chewing paan and aspects of dental and periodontal status were found. Impacts of oral health were reported by 45% of the respondents. The normative need for dental treatment was 96% with a perceived need of 48%. CONCLUSION: There was considerable normative dental need. Whilst caries experience was low, there were high levels of periodontal treatment needs. Paan chewing was related to aspects of dental and periodontal status.

Prevalence of oral lesions among a sample of Bangladeshi medical users aged 40 years and over living in Tower Hamlets, UK.

AIM: To collect data on the prevalence of oral mucosal lesions among Bangladeshi medical care users aged 40 years and over and to explore the relationship of oral disease status and tobacco smoking and paan (betel quid) chewing. DESIGN: Multi-centre cross-sectional study. SETTING: General medical practitioners' waiting areas in Tower Hamlets, UK. SUBJECTS: 185 Bangladeshi adults aged 40 years old and over. METHOD: An oral mucosal examination, based on WHO criteria and an interview. Information on tobacco smoking and paan chewing behaviour was collected during the interview. RESULTS: Out of 185 adults there was a response rate of 74%. Oral mucosal lesions were observed in 40% of participants. The most common lesion was found to be leukoplakia with a prevalence of 25%. Significant relationships were found between smoking and the presence of oral pathology and between paan chewing with tobacco and the presence of leukoplakia. CONCLUSION: In this study of older Bangladeshi medical care users there was a high prevalence of oral mucosal lesions. Sensitively tailored health promotion interventions, which aim to reduce tobacco use, should be developed for this population.

The Th2 cytokine environment of the placenta.

It is now accepted that local changes to the balance of Th1/Th2-type cytokines occur during pregnancy within the maternal uterus and fetoplacental unit. These changes in cytokine profiles contribute to implantation of the embryo, development of the placenta, and survival of the fetus to term. Overall within the placenta there is a bias in the ratio of Th1:Th2 cytokines towards the Th2-type cytokines. However, there are specific fluctuations in this balance at implantation and during the initiation of parturition. The predominant cytokines at each stage of gestation function both to limit maternal immune rejection of the semi-allogeneic embryo/fetus, especially at the maternofetal interface; and to facilitate the on-going physiological processes within the maternal reproductive tract. These two, at times conflicting, roles are discussed in this review, with key evidence concerning cytokine expression and function from mouse and humans.

Mobilizing store Ca(2+) in the presence of La(3+) evokes exocytosis in bovine chromaffin cells.

The effect on exocytosis of La(3+), a known inhibitor of plasma membrane Ca(2+)-ATPases and Na(+)/Ca(2+) exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3-3 mM), La(3+) substantially increased histamine-induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd(3+), Eu(3+), Gd(3+), and Tb(3+)), but not several divalent cations. In the presence of La(3+), the secretory response to histamine became independent of extracellular Ca(2+). La(3+) enhanced secretion evoked by other agents that mobilize intracellular Ca(2+) stores (angiotensin II, bradykinin, caffeine, and thapsigargin), but not that due to passive depolarization with 20 mM K(+). La(3+) still enhanced histamine-induced secretion in the presence of the nonselective inhibitors of Ca(2+)-permeant channels SKF96365 and Cd(2+), but the enhancement was abolished by prior depletion of intracellular Ca(2+) stores with thapsigargin. La(3+) inhibited (45)Ca(2+) efflux from preloaded chromaffin cells in the presence or absence of Na(+). It also enhanced and prolonged the rise in cytosolic [Ca(2+)] measured with fura-2 during mobilization of intracellular Ca(2+) stores with histamine in Ca(2+)-free buffer. The results suggest that the efficacy of intracellular Ca(2+) stores in evoking exocytosis is enhanced dramatically by inhibiting Ca(2+) efflux from the cell.

Differential control of tyrosine hydroxylase activation and catecholamine secretion by voltage-operated Ca2+ channels in bovine chromaffin cells.

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.

Different contributions of voltage-sensitive Ca2+ channels to histamine-induced catecholamine release and tyrosine hydroxylase activation in bovine adrenal chromaffin cells.

Histamine stimulates catecholamine release and tyrosine hydroxylase activity in a Ca(2+)-dependent manner in bovine adrenal chromaffin cells. The role of voltage-sensitive Ca2+ channels in these two responses has been investigated. Using an EC50 concentration of histamine, 1 microM, catecholamine release was enhanced by (+/-)BayK8644, and partially inhibited by nitrendipine and omega-agatoxin IVA, blockers of L- and P/Q-type Ca2+ channels. omega-Conotoxin GVIA gave small and variable inhibitory effects. With a maximal histamine concentration, 10 microM, similar results were obtained except that now omega-conotoxin GVIA reliably reduced release. In contrast, neither (+/-)BayK8644 nor any of the individual Ca2+ channel antagonists had any significant effect on tyrosine hydroxylase (TOH) activation induced by either an EC50 or a maximal concentration of histamine. When high concentrations of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA were combined with omega-conotoxin MVIIC (a non-selective blocker of N, P and Q channels) to block voltage-sensitive Ca2+ channels in these cells, release induced by K+ depolarization was completely blocked. Release caused by histamine, however, was substantially reduced but not abolished. The combination of antagonists also only partially inhibited TOH activation by histamine. The results show that the G protein-coupled receptor agonist histamine activates several different types of voltage-sensitive Ca2+ channels in chromaffin cells to mediate its cellular effects. Histamine may also activate additional pathways for Ca2+ entry. The results also suggest that the manner by which Ca2+ controls release and TOH activation once it has entered chromaffin cells through these channels are different.

Dental service use and the implications for oral cancer screening in a sample of Bangladeshi adult medical care users living in Tower Hamlets, UK.

AIM: To assess the use of dental services, barriers to uptake of dental care and attitudes to regular dental examinations and the prevalence of tobacco and paan chewing habits in a group of Bangladeshi medical care users. DESIGN: Multi-centre cross-sectional study. SETTING: Four general medical practices' waiting areas in Tower Hamlets. SUBJECTS: Bangladeshi adults aged 40 years and over. INTERVENTION: An interview schedule. MAIN OUTCOME MEASURES: The prevalence of tobacco smoking and paan chewing with or without the addition of tobacco. The use of dental services, barriers to the use of dental services and attitudes to regular dental examinations. RESULTS: Results were obtained from 158 subjects (response rate 85%). 25% of the whole sample had never visited a dentist. These were significantly (P < 0.05) more likely to be women, who also thought regular check-ups were of little value. In their use of health services 73% experienced language difficulties. 33% of the sample were tobacco smokers. Paan was chewed by 78% of the sample with significantly (P < 0.05) more females than males adding tobacco to their quid and chewing more frequently than males. CONCLUSION: There are considerable barriers to be overcome if dental practices are to be the site for oral cancer screening and oral health promotion in this population. There are sex differences in reported behaviour and attitudes about use of dental services and in tobacco and paan use in this Bangladeshi sample. Further research is needed to establish why this ethnic minority attend general medical practices but not general dental practices.

The prevalence of dental erosion in the maxillary incisors of 14-year-old schoolchildren living in Tower Hamlets and Hackney, London, UK.

The purpose of this study was to assess the prevalence of dental erosion in the maxillary incisors of a sample of 14-year-old schoolchildren and to explore the aetiological factors responsible for that erosion. The cross-sectionally design study took place in secondary schools in inner-city London, UK and involved 525, 14-year-old schoolchildren selected at random in a clinical examination and a self-completed questionnaire. The outcomes measures for dental erosion were; the prevalence, the area and depth of lesions and the risk factors. The prevalence of labial and palatal erosion was 16.9 per cent and 12 per cent respectively. Risk factors and behaviours including daily frequency of ingestion of acidic fruits and drinks, food vomiting, toothbrushing frequency, and swimming habits were not shown to have any relationship with the presence of erosion. It was concluded that the prevalence of erosion in the maxillary incisors of this sample was higher labially and lower palatally than in previously reported national figures. The risk factors which were investigated were not shown to have any relationship with the presence of erosion. Further investigations of these issues are necessary to establish whether or not dental erosion is a public health problem in the UK.

Higher plants possess two structurally different poly(ADP-ribose) polymerases.

One of the immediate reactions of the mammalian cell to many environmental stresses is a massive synthesis of poly(ADP-ribose), catalyzed by poly(ADP-ribose) polymerase (PARP). Most of the biological functions attributed to PARP are inferred from experimentation with mammalian cells. In plants, the biology of PARP may be more complicated and diverse than was previously thought. Two poly(ADP-ribose) polymerase homologues were found in plants, the classical Zn-finger-containing polymerase (ZAP) and the structurally non-classical PARP proteins (APP and NAP), which lack the characteristic N-terminal Zn-finger domain. By enzymatic and cytological experiments the recombinant APP protein was shown to be located in the nucleus and to possess DNA-dependent poly(ADP-ribose) polymerase activity in yeast. The nuclear localization was further confirmed by the analysis of transgenic tobacco plants that expressed a translational gene fusion between APP and the bacterial beta-glucuronidase. The app promoter was transcriptionally up-regulated in cells pre-determined to die because of deficiency in a DNA ligase I.

Multiple calcium channels are required for pituitary adenylate cyclase-activating polypeptide-induced catecholamine secretion from bovine cultured adrenal chromaffin cells.

The effects of L-, N-, P- and Q-type calcium channel antagonists and (+/-)-BayK-8644 on catecholamine release induced by pituitary adenylate cyclase-activating polypeptide (PACAP-27) were investigated in bovine cultured adrenal chromaffin cells. PACAP-27 induced the release of 4-15% of the total cellular catecholamines over 7 min, with an EC50 of 20 nM and the effect approaching maximum at 100 nM. Catecholamine release was fully dependent on the presence of extracellular calcium. The dihydropyridine nitrendipine which inhibits L-type calcium channels inhibited PACAP-27-induced secretion in a concentration dependent manner with an inhibition of 20-30% at 1 microM. In contrast, (+/-)-BayK-8644, which prolongs the opening of L-type calcium channels produced a concentration-dependent increase in PACAP-27-induced catecholamine release with 1 microM increasing release by 40-60%. Blockade of N-type calcium channels with omega-conotoxin GVIA reduced release by 5-15%. Block of P-type channels with low concentrations of omega-agatoxin IVA (< or = 30 nM) had no significant effect on release, while higher concentrations (100-300 nM) which block Q-type channels reduced release by up to 15%. omega-Conotoxin MVIIC, an antagonist of Q-type calcium channels and also of N- and P-type channels, inhibited release in a concentration-dependent manner with a near maximum effect of 30-50% produced by 300 nM. The combination of omega-conotoxin GVIA and omega-agatoxin IVA reduced release by 40-50%. Addition of omega-conotoxin MVIIC (300 nM) to the combination of omega-conotoxin GVIA (10 nM) and omega-agatoxin IVA (100 nM) did not inhibit catecholamine release more than with omega-conotoxin GVIA and omega-agatoxin IVA alone, indicating that 100 nM omega-agatoxin IVA was sufficient to block the Q-type calcium channels. When nitrendipine was used together with omega-conotoxin GVIA, omega-agatoxin IVA and omega-conotoxin MVIIC, catecholamine release induced by 20 nM or 100 nM PACAP-27 was reduced by 70-85%. Taken together these results suggest that influx of calcium through multiple different voltage-sensitive calcium channels mediate PACAP-27-induced catecholamine release from bovine chromaffin cells, and that L-, N- and Q-channels contribute to this response.

Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching.

Poly(ADP-ribose) polymerase (PARP) is a multifunctional nuclear zinc finger protein which participates in the immediate response of mammalian cells exposed to DNA damaging agents. Given the complexity of the poly(ADP-ribosylation) reaction, we developed a large-scale screening procedure in Escherichia coli to identify randomly amino acids involved in the various aspects of this mechanism. Random mutations were generated by the polymerase chain reaction in a cDNA sequence covering most of the catalytic domain. Out of 26 individual mutations that diversely inactivated the full-length PARP, 22 were found at conserved positions in the primary structure, and 24 were located in the core domain formed by two beta-sheets containing the active site. Most of the PARP mutants were altered in poly(ADP-ribose) elongation and/or branching. The spatial proximity of some residues involved in chain elongation (E988) and branching (Y986) suggests a proximity or a superposition of these two catalytic sites. Other residues affected in branching were located at the surface of the molecule (R847, E923, G972), indicating that protein-protein contacts are necessary for optimal polymer branching. This screening procedure provides a simple and efficient method to explore further the structure-function relationship of the enzyme.

Effects of N- and L-type calcium channel antagonists and (+/-)-Bay K8644 on nerve-induced catecholamine secretion from bovine perfused adrenal glands.

1. The effects of N- and L-type calcium channel antagonists and (+/-)-Bay K8644 on catecholamine release from chromaffin cells and acetylcholine release from splanchnic nerve terminals was investigated in bovine perfused adrenal glands. 2. Adrenal glands were perfused retrogradely and preloaded with [3H]-choline. Subsequent efflux of 3H-labelled compounds was taken as an index of acetylcholine release from the splanchnic nerve terminals. Noradrenaline and adrenaline release from the glands was measured by h.p.l.c. with electrochemical detection. 3. A maximally effective frequency of field stimulation of the adrenal nerves, 10 Hz, induced release of catecholamines and 3H-labelled compounds. Tetrodotoxin (1 microM) abolished release of both catecholamines and 3H-labelled compounds. A combination of mecamylamine (5 microM) and atropine (1 microM) inhibited nerve-induced catecholamine release by about 75% but did not inhibit release of 3H-labelled compounds. Reducing the concentration of extracellular calcium 5 fold to 0.5 mM inhibited nerve-induced catecholamine release by 80% and release of 3H-labelled compounds by 50%. 4. (+/-)-Bay K8644 (1 microM), nitrendipine (1 microM), omega-conotoxin-GVIA (10 nM) and the combination of nitrendipine and omega-conotoxin-GVIA each had no effect on nerve-induced release of 3H-labelled compounds. 5. (+/-)-Bay K8644 (1 microM) potentiated nerve-induced catecholamine release by 75%. Nitrendipine (1 microM) reduced release by 20% but this did not reach statistical significance, omega-Conotoxin-GVIA (10 nM) reduced nerve-induced catecholamine release by 75%, while the combination of omega-conotoxin-GVIA and nitrendipine reduced release to the same extent as omega-conotoxin-GVIA alone. 6. Exogenous acetylcholine perfusion through the glands produced a concentration-dependent increase in catecholamine release. The maximally effective concentration of acetylcholine for catecholamine release was > or = 300 microM, while 30 microM acetylcholine gave comparable catecholamine release to that obtained with 10 Hz field stimulation. 7. (+/-)-Bay K8644 (1 microM), nitrendipine (1 microM) and omega-conotoxin-GVIA (10 nM) each had no significant effect on catecholamine release evoked by perfusion of the gland with either a near maximally effective concentration of acetylcholine, 100 microM, or with the lower concentration of 30 microM. 8. The results show that the omega-conotoxin-GVIA-sensitive N-type voltage-sensitive calcium channels located on the chromaffin cells are largely responsible for catecholamine release induced by nerve stimulation in bovine adrenal glands. In contrast, N-type calcium channels are not involved in catecholamine release induced by exogenous acetylcholine. L-type voltage sensitive calcium channels do not play a major role in nerve-induced or exogenously applied acetylcholine-induced catecholamine release. However, the L-type calcium channels do have the potential to augment powerfully nerve-induced catecholamine release. N- and L-type calcium channels do not play a major role in the presynaptic release of acetylcholine.

Caffeine enhances sympathetic purinergic and noradrenergic transmission in the guinea-pig isolated vas deferens.

Intracellular recording techniques were used to monitor the resting membrane potential of smooth muscle cells and the excitatory junction potentials (EJPs) evoked by stimulation of the hypogastric nerve. Stimulation with trains of 15 pulses at 1 Hz or 0.33 Hz evoked individual EJPs which increased in amplitude from the first pulse and reached a plateau after 6-8 pulses. Stimulation at 1 Hz resulted in EJPs facilitating to a plateau level of approximately 25 mV, whereas with stimulation at 0.33 Hz the EJPs only facilitated to a plateau level of about 12 mV. With stimulation at 1 Hz, caffeine (3 mM and 10 mM), increased the amplitude of the first few EJPs in each train and decreased the extent of facilitation and reduced the amplitude of fully facilitated EJPs. In comparison, the amplitude of all EJPs evoked by stimulation at 0.33 Hz was increased by caffeine (3 mM and 10 mM). With 0.33 Hz stimulation, facilitation of the first few EJPs was observed in the presence of 3 mM caffeine but not in the presence of 10 mM caffeine. In the presence of the alpha 2-adrenoceptor antagonist idazoxan, caffeine (3 mM and 10 mM) still enhanced the amplitude of EJPs early in trains of stimulation but there was no depression of EJPs later in the trains. Similarly, in reserpine-treated vasa deferentia, caffeine (3 mM) enhanced EJPs early in the train of stimulation at 1 Hz and there was no depression of EJPs at the end of the train. In addition to electrophysiological experiments, the effect of caffeine (0.1-30 mM) on the resting and stimulation-induced (S-I) efflux of radioactivity was investigated in guinea-pig isolated vasa deferentia previously incubated with [3H]-noradrenaline. Caffeine (10 mM) did not affect the resting efflux of [3H]-noradrenaline but significantly enhanced the S-I efflux by 150-160%. The present findings suggest that caffeine enhances sympathetic purinergic and noradrenergic transmission at the sympathetic neuroeffector junction in the guinea-pig vas deferens. Moreover, the increased release of transmitter noradrenaline can modulate purinergic transmission by activation of alpha 2-adrenoceptors located at sympathetic neuroeffector sites.

Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase.

In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described. Here, we have used mutagenesis with hydroxylamine to generate a random library of PARP mutants. The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F) is described. We show that the kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged. The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme. This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage.