Attitudes and behaviour of ophthalmologists to smoking cessation.

AIMS: Growing evidence suggests a causal association between smoking and eye disease. This study explores the current beliefs and practice among UK consultant ophthalmologists towards delivering smoking cessation advice to eye clinic attenders. METHODS: A cross-sectional survey using a postal questionnaire of all UK NHS hospital based consultant ophthalmologists was conducted. The questionnaire explored whether: ophthalmologists identify the smoking status of their patients, advise about the increased risk of eye diseases among smokers, and deliver smoking cessation advice. The availability of departmental smoking cessation resources was also ascertained. RESULTS: The response rate was 55% (485/886). Of the responders 79% were males. Only 35% of responders asked about smoking status every time or most times for new patients and 5% for follow-up patients. In all, 40% claimed to always or usually advise patients to quit smoking and 61% claimed to always or usually mention eye disease as a reason to quit. Only 14% assessed motivation to quit and 22% provided advice and assistance about how to stop smoking to smokers who wished to quit. Female ophthalmologists were more likely to undertake most aspects of smoking assessment and intervention. Only 18% of responders stated that their departments provide information about smoking for patients and 6% stated that support is available for patients wanting to quit smoking. CONCLUSION: The assessment of smoking status and provision of targeted support for smokers to quit could be substantially improved in UK ophthalmology departments. There is a need to introduce smoking cessation support into routine ophthalmic practice and provide the resources to support this.

Attitudes of community optometrists to smoking cessation: an untapped opportunity overlooked?

AIM: To assess community optometrists' attitudes and current behaviour regarding provision of smoking cessation advice in their practice. METHODS: A self-completion postal questionnaire was sent to community optometrists in north-west England identified from the General Optical Council's practice lists. RESULTS: Of 709 optometrists identified, 71.8% (509/709) returned the completed questionnaire. Few community optometrists routinely asked about smoking habits: only 6.2% (95% CI: 4.1-8.3) (n = 31) at new patient consultations, and 2.2% (95% CI: 0.9-3.5) (n=11) at follow-up visits. Reasons for optometrists not routinely providing smoking cessation advice included: not their role (35.4%, n=180), lack of time (22.0%, n=112) and forgetting to ask (21.4%, n=109). Overall 67.6% (95% CI: 63.5-71.7) (n=344) of community optometrists wanted to improve their knowledge of smoking and visual impairment with 56.2% (95% CI: 51.9-60.5) (n=286) requesting further training. CONCLUSION: Despite low levels of current involvement, many optometrists were keen to receive training on smoking cessation topics. We suggest that there are untapped opportunities to develop brief interventions to promote smoking cessation services in community optometry settings.

Effects of bromodichloromethane on ex vivo and in vitro luteal function and bromodichloromethane tissue dosimetry in the pregnant F344 rat.

Bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss, i.e. full-litter resorption, in F344 rats when treated during the luteinizing hormone (LH)-dependent period. This effect is associated with reduced maternal serum progesterone (P) and LH levels, suggesting that BDCM disrupts secretion of LH. To test the hypothesis that BDCM also affects luteal responsiveness to LH, we used ex vivo and in vitro approaches. For the ex vivo study (i.e., in vivo exposure followed by in vitro assessment), dams were dosed by gavage on gestation days (GD) 6-9 (plug day=GD 0) at 0 or 100 mg/kg/d. One hour after the GD-9 dose, rats were killed, blood was collected, and tissue concentrations of BDCM were assessed. Corpora lutea (CL) were incubated with or without hCG, an LH agonist, to stimulate P secretion. For the in vitro study, CL were pooled from untreated F344 rats on GD 9 and cultured with BDCM at 0, 0.01, 0.10 or 3.0 mM. BDCM was found at highest concentrations in adrenal, ovarian, adipose, and hypothalamic tissues. BDCM treatment decreased serum P and LH levels in vivo. Ex vivo, however, BDCM-exposed CL showed >2-fold increases in P secretion relative to controls. Both control and BDCM-exposed CL displayed a 2.4-fold increase in P secretion in response to hCG challenge. In contrast, in vitro exposures reduced CL responsiveness in a dose-related fashion while baseline levels were unaffected. It is unclear if the ex vivo 'rebound' reflects the removal of the CL from a possible direct inhibitory influence of BDCM, or a response to diminished LH stimulation in vivo. Thus, these data suggest that BDCM disrupts pregnancy in F344 rats via two modes: disruption of LH secretion, and disruption of the CL's ability to respond to LH.

Signalling pathways involved in the control of sperm cell volume.

The ability to maintain cellular volume is an important general physiological function, which is achieved by specific molecular mechanisms. Hypotonically induced swelling results in the opening of K+ and Cl- ion channels, through which these ions exit with accompanying water loss. This process is known as regulatory volume decrease (RVD). The molecular mechanisms that control the opening of the ion channels in spermatozoa are as yet poorly understood. The present study investigated pathways of osmo-signalling using boar spermatozoa as a model. Spermatozoa were diluted into isotonic and hypotonic Hepes-buffered saline in the presence or absence of effector drugs, and at predetermined intervals volume measurements were performed electronically. Treatment with protein kinase C (PKC) inhibitors staurosporine, bismaleimide I and bismaleimide X led to dose-dependent increases of both isotonic and hypotonic volumes (P<0.05). However, as the isotonic volume was affected more than the hypotonic volume, the kinase inhibitors appeared to improve RVD, whereas activation of PKC with phorbol dibutyrate blocked RVD. The increase in isotonic cell volume induced by bismaleimide X was observed in chloride-containing medium but not in the medium in which chloride was replaced by sulphate, implying that PKC was involved in the control of chloride channel activity, e.g. by closing the channel after volume adjustment. The protein phosphatase PP1/PP2 inhibitors calyculin and okadaic acid increased the isotonic volume only slightly but they greatly increased the relative cell volume and blocked RVD. The activation of RVD processes was found to be cAMP-dependent; incubation with forskolin and papaverine improved volume regulation. Moreover, papaverine was able to overcome the negative effect of protein phosphatase inhibitors. The mechanism of sperm RVD appears to involve (a) alterations in protein phosphorylation/dephosphorylation balance brought about by PKC and PP1 and (b) a cAMP-dependent activating pathway.

Cigarette smoking and thyroid eye disease: a systematic review.

PURPOSE: To evaluate the epidemiological evidence for a causal association between tobacco smoking and thyroid eye disease (TED). METHODS: Systematic review, including quality assessment, of published epidemiological studies and evaluation of the evidence using established causality criteria. RESULTS: Fourteen papers describing 15 studies were included. There was a positive association between smoking and TED in four case-control studies when compared with control patients with Graves' disease but no ophthalmopathy (odds ratio (OR) 1.94-10.1) and in seven case-control studies in which control subjects did not have thyroid disease (OR 1.22-20.2). Two cohort studies examined the occurrence of new cases of TED; one study found an increased incidence of TED with smoking. Four cohort studies investigated progression or outcome of treatment in patients with established TED, three finding an association between smoking and poorer outcome. The quality of the studies was variable, but the association with smoking was strong in the most methodologically rigorous studies. Other evidence supporting a causal link was a consistent association across studies, a dose-response effect, a reduced risk of TED in ex-smokers, and a temporal relationship. CONCLUSION: This systematic review provided strong evidence for a causal association between smoking and development of TED. Current-smokers were also more likely to experience disease progression or poorer outcome of treatment. Patients with Graves' disease and the general public should be educated about the risk of smoking and TED. These findings suggest that patients with Graves' disease or TED who are smokers should be given effective support to stop smoking.

Pleasure or pain? A profile of smokers in Northern England.

OBJECTIVE: To construct a profile of smokers using multiple indices of physical, mental and social health. STUDY DESIGN: Cross-sectional study. METHODS: The setting was Wigan and Bolton Health Authority, an urban district in the North West of England. A random sample of over 15000 adults from the Health Authority adult population completed a written questionnaire. Prevalence ratios were calculated for physical, mental and social health indicators for smokers compared with non-smokers, adjusted for borough, age and deprivation score of place of residence. RESULTS: Smokers were less likely to report their current health as good, and reported a significantly higher prevalence of arthritis, bronchitis, backache and respiratory symptoms. Smokers had more mobility problems and recent severe pain. Smokers had less healthy lifestyles across many behaviours (e.g. poorer diet, taking less regular exercise and more problem drinking). Depression and the proportion of people with a high psychiatric morbidity score were increased. More women smokers reported a lack of social support, and smokers more often reported financial difficulties. Differences were exaggerated by comparing heavy smokers with non-smokers. CONCLUSIONS: Independent of the level of deprivation of their area of residence, smokers have poorer physical, social and mental health, with a dose-response effect. Smoking creates considerable pain, but little evidence of pleasure.

Can pathological complete response in the primary tumour following pre-operative pelvic chemoradiotherapy for T3-T4 rectal cancer predict for sterilisation of pelvic lymph nodes, a low risk of local recurrence and the appropriateness of local excision?

INTRODUCTION: Local excision is considered inappropriate treatment for T3-T4 rectal adenocarcinomas, as it cannot provide prognostic information regarding lymph node involvement and has a high risk of pelvic recurrence. Preoperative chemoradiation (CRT) studies in rectal cancer suggest that a pathological complete response (pCR) in the primary tumour provides an excellent long-term outcome. If downstaging to stage pT0 predicts a tumour response within the perirectal and pelvic lymph nodes, this may allow local excision to be performed without increased risk of pelvic recurrence. This retrospective study aimed to determine the incidence of involved lymph nodes following pCR (ypT0) after preoperative CRT and total mesorectal excision. METHOD: The outcome and treatment details of 211 patients undergoing preoperative CRT for clinically staged T3-T4 unresectable rectal adenocarcinomas between 1993 and 2003 at Mount Vernon Hospital were reviewed. RESULTS: Data were recorded from the 143 patients who completed treatment with a median follow-up of 25 months. Twenty-three patients (18%) were found to have had a pCR. Four out of 23 patients (17%) had involved lymph nodes. No pelvic recurrences developed after a ypCR. Overall survival was similar for patients with ypT0 or residual tumour. CONCLUSION: Pathological complete response in the primary tumour failed to predict a response in the perirectal lymph nodes (p=0.08). The degree of response predicted a lymph node response (p=0.02). The detection of ypCR identified patients with a low rate of pelvic recurrence. This may in the future allow selection of patients for whom local excision can be performed without a higher risk of local relapse.

Planning to win: Health and lifestyles associated with physical activity amongst 15,423 adults.

STUDY OBJECTIVE: To assess levels of physical activity in the general population and amongst the 'healthy', and to identify factors associated with this important health behaviour. DESIGN: Population-based cross-sectional study. SETTING: North-west England. PARTICIPANTS: Adults (aged at least 18 years) registered with a general practitioner with a residential address within two local administrative districts (local authorities). MAIN RESULTS: Less than one-third of adults performed adequate amounts of physical activity for health protection, and this differed little when analyses were restricted to 'healthy' people. Lower levels of physical activity were observed amongst women, older people, ethnic groups, those with obesity and in each increased quintile of social deprivation. Current smokers, but not previous smokers, were less likely to be physically active, as were those not eating at least five portions of fruit and vegetables per day. Lack of physical activity was associated with poor general health and a history of, or current, chronic disease. CONCLUSIONS: Concerted efforts are required by the Government, society and individuals to overturn our predominantly physically inactive adult population. Interventions may be needed which specifically target certain groups, especially the most socially deprived, and that consider individuals and societal barriers to becoming physically active. Evidence of the effectiveness of individual and population-based interventions remains scant and this needs to be addressed urgently.

Smoking and age-related macular degeneration: a review of association.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of severe and irreversible vision loss in the Western world. As there is no effective treatment for all types of AMD, identifying modifiable risk factors is of great importance. This review evaluates the epidemiological evidence associating smoking with AMD. METHODS: Systematic review of published epidemiological studies evaluated against established criteria for evidence of a causal relationship. RESULTS: In total, 17 studies (cross-sectional studies, prospective cohort studies, and case-control studies) were included in the review. A total of 13 studies found a statistically significant association between smoking and AMD with increased risk of AMD of two- to three-fold in current-smokers compared with never-smokers. Five studies found no association between smoking and AMD. There was also evidence of dose-response, a temporal relationship and reversibility of effect. CONCLUSION: The literature review confirmed a strong association between current smoking and AMD, which fulfilled established causality criteria. Cigarette smoking is likely to have toxic effects on the retina. In spite of the strength of this evidence, there appears to be a lack of awareness about the risks of developing eye disease from smoking among both healthcare professionals and the general public.

Perceptions of blindness related to smoking: a hospital-based cross-sectional study.

AIMS: Smoking is associated with several serious eye diseases. Awareness of smoking and blindness, and its potential to act, as a stimulus to assist stopping smoking has not been investigated. METHODS: A cross-sectional survey using a structured interview of adult patients attending district general hospital ophthalmology, general surgery, and orthopaedic clinics. The interview investigated the awareness and fear of blindness for three established smoking-related diseases, and a distractor condition (deafness), and the likelihood that smokers would quit on developing early signs of each condition. RESULTS: Response was 89.1% (358/402). In all, 183 (51.1%) of responders were male and 175 (48.9%) female. Only 9.5% of patients believed that smoking was definitely or probably a cause of blindness, compared with 92.2% for lung cancer, 87.6% for heart disease, and 70.6% for stroke. Patients ranked their fear of each of the five conditions, scoring five for the most feared and one for the least feared. Patients were significantly (P<0.01) less fearful of blindness (mean score 2.80) than lung cancer (3.89), heart disease (3.58), and stroke (3.35). About one-half of smokers stated that they would definitely or probably quit smoking if they developed early signs of blindness or the three established smoking-related conditions, with no significant differences in proportions for these four conditions. CONCLUSION: The findings suggest that awareness of the risk of blindness from smoking is low, but that the fear of blindness is as compelling a motivation to quit as fear of lung cancer, heart disease, and stroke. The link between smoking and eye disease should be publicised to help reduce smoking prevalence.

The reliability of lymph-node staging in rectal cancer after preoperative chemoradiotherapy.

AIMS: To determine the prognostic significance of the nodal stage and number of nodes recovered in the surgical specimen after preoperative synchronous chemoradiation (SCRT) and surgery for locally advanced or unresectable rectal cancer. MATERIALS AND METHODS: One hundred and eighty-two consecutive patients with locally advanced or unresectable (T3/T4) rectal carcinomas were entered on a prospective database and treated in this department with preoperative chemoradiation, followed 6-12 weeks later by surgical resection. Most patients received chemotherapy in the form of low-dose folinic acid and 5-fluorouracil (5-FU) 350 mg/m2 via a 60-min infusion on days 1-5 and 29-33 of a course of pelvic radiotherapy delivered at a dose of 45 Gy in 25 fractions over 33 days to a planned volume. After resection, patients with a positive circumferential margin (< or = 1 mm), extranodal deposits or Dukes' C histology received adjuvant 5-FU-based-chemotherapy (n = 40). RESULTS: After SCRT, 161 patients underwent resection. Twenty-one patients remained unresectable or refused an exenterative operation. Median follow-up is 36 months. Down-staging was achieved in most patients, with 19 having a complete pathological response (pT0). The median number of lymph nodes recovered for all patients was five (range 0-21). The 3-year survival rate for node-positive patients is 47%, for node-negative patients with less than three lymph nodes recovered is 62% and for node-negative patients with three or more lymph nodes recovered is 70%. Compared with node-positive patients, simple regression models revealed a reduced hazard ratio (HR) of 0.72 (0.36-1.43) for node-negative patients with less than three nodes recovered and 0.48 (0.26-0.89) for node-negative patients with three or more lymph nodes recovered. In a multivariate model, including nodal status, excision status, age and sex only positive excision margins significantly predicted a poor outcome: HR = 3.05 (1.55-5.97). CONCLUSIONS: The number of nodes found after preoperative chemoradiation is a significant prognostic factor by univariate analysis. In this study, patients with node-negative histology, and at least three nodes recovered, had better long-term survival than patients in whom two or less nodes were recovered or with positive nodes. This effect was attenuated by the inclusion of excision status in multivariate models.

Is there a role for long-chain omega3 or oil-rich fish in the treatment of atrial fibrillation?

Atrial fibrillation is the most common cardiac arrhythmia in Europe and north America, and recently it was described as an epidemic. Treatment and management of this arrhythmia consists of using drugs, external electrical cardioversion and in extreme cases, internal electrical pacing. Despite treatment, this arrhythmia continues to impact on morbidity and mortality. The possible benefit from dietary interventions in relation to the primary and secondary prevention of atrial fibrillation have largely been overlooked. Our hypothesis is that increasing the intake of long-chain polyunsaturated omega3 fatty acids (LCn3) from eating a diet containing moderate amounts of oil-rich fish, will benefit people with persistent atrial fibrillation. A number of possible anti-arrhythmic actions from LCn3 have been found from animal and laboratory studies, mainly on ventricular arrhythmias. These include reducing pro-arrhythmic eicosanoids and inhibiting sodium and calcium currents. If found to be beneficial to these patients, dietary advice to eat more oil-rich fish, or take LCn3 supplements, could be part of a package of care for people with this arrhythmia. We have currently started a randomised controlled trial to test our hypothesis.

Omega 3 fatty acids for prevention and treatment of cardiovascular disease.

BACKGROUND: It has been suggested that omega 3 (W3, n-3 or omega-3) fats from oily fish and plants are beneficial to health. OBJECTIVES: To assess whether dietary or supplemental omega 3 fatty acids alter total mortality, cardiovascular events or cancers using both RCT and cohort studies. SEARCH STRATEGY: Five databases including CENTRAL, MEDLINE and EMBASE were searched to February 2002. No language restrictions were applied. Bibliographies were checked and authors contacted. SELECTION CRITERIA: RCTs were included where omega 3 intake or advice was randomly allocated and unconfounded, and study duration was at least six months. Cohorts were included where a cohort was followed up for at least six months and omega 3 intake estimated. DATA COLLECTION AND ANALYSIS: Studies were assessed for inclusion, data extracted and quality assessed independently in duplicate. Random effects meta-analysis was performed separately for RCT and cohort data. MAIN RESULTS: Forty eight randomised controlled trials (36,913 participants) and 41 cohort analyses were included. Pooled trial results did not show a reduction in the risk of total mortality or combined cardiovascular events in those taking additional omega 3 fats (with significant statistical heterogeneity). Sensitivity analysis, retaining only studies at low risk of bias, reduced heterogeneity and again suggested no significant effect of omega 3 fats. Restricting analysis to trials increasing fish-based omega 3 fats, or those increasing short chain omega 3s, did not suggest significant effects on mortality or cardiovascular events in either group. Subgroup analysis by dietary advice or supplementation, baseline risk of CVD or omega 3 dose suggested no clear effects of these factors on primary outcomes. Neither RCTs nor cohorts suggested increased relative risk of cancers with higher omega 3 intake but estimates were imprecise so a clinically important effect could not be excluded. REVIEWERS' CONCLUSIONS: It is not clear that dietary or supplemental omega 3 fats alter total mortality, combined cardiovascular events or cancers in people with, or at high risk of, cardiovascular disease or in the general population. There is no evidence we should advise people to stop taking rich sources of omega 3 fats, but further high quality trials are needed to confirm suggestions of a protective effect of omega 3 fats on cardiovascular health. There is no clear evidence that omega 3 fats differ in effectiveness according to fish or plant sources, dietary or supplemental sources, dose or presence of placebo.

Are those in need taking dietary supplements? A survey of 21 923 adults.

Many people take dietary supplements, but information on characteristics associated with their use is lacking. The relationship between lifestyle behaviours, morbidity and use of dietary supplements has not been examined and earlier studies have limited applicability to a general population. These issues were addressed in the current study. Information was obtained by postal questionnaire sent to a sample of the general population. The questionnaire was completed by 70.5 % of the sample (15 465 from a total sample of 21 923), with at least one-third (35.5 %) taking dietary supplements. In adjusted analyses, supplement users were more likely to be women, white, home-owners, non-smokers and physically active. Use of vitamin, mineral and/or antioxidant supplements was associated with eating more fruits and vegetables, and taking fish-oil supplements was associated with eating oil-rich fish. A history of CVD or risk factors for CVD reduced the risk of taking vitamins, minerals and/or antioxidants or fish-oil supplements. Those reporting musculoskeletal disorders such as arthritis were more likely to take fish-oil supplements For the first time, we have shown that dietary supplement use is related to different types of morbidity. In particular, people at risk of primary or secondary CVD seem less likely to use dietary supplements, despite possible benefits shown in clinical trials. Public health organisations need to develop guidelines for the public and health professionals regarding the uncontrolled use of dietary supplements in the community.

Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate.

In boar spermatozoa, the capacitating agent bicarbonate has been shown to induce rapid changes both in plasma membrane lipid architecture and in motility; in each case, a PKA-dependent pathway is involved. Early bicarbonate-induced changes in protein phosphorylation were probed using a commercial antibody against the phosphorylated form of the consensus substrate site for cyclic AMP-dependent protein kinase. The antibody detected relatively few bands in sperm extracts, of which only a small number showed incubation-dependent changes. While the quantitative response varied between boar ejaculates, in general terms bicarbonate induced phosphorylation increases in bands of 96, 64, and 59 kDa within 80 sec. The changes reached a maximum after about 160 sec, declined somewhat thereafter, and then increased again slowly as incubation progressed further (up to 21 min). The bicarbonate-induced increases were strongly dependent on the presence of BSA in the incubation medium. They were inhibited by H89 (PKA inhibitor) but not by GF (PKC inhibitor), and were enhanced by papaverine (phosphodiesterase inhibitor) and by calyculin (protein phosphatase inhibitor). The cyclic AMP analogue cBIMPS was able to mimic bicarbonate action though its effect was less dramatic. Stearated Ht31, a permeable inhibitor of PKA's binding to A-kinase anchoring protein, did not affect either the intensity or the specificity of the bicarbonate-induced phosphorylation changes, though it blocked motility entirely. Immunocytochemical studies revealed marked bicarbonate-dependent phosphorylation changes in the post-acrosomal region of the head and in the neck, midpiece, and anterior regions of the tail. Fractionation of stimulated spermatozoa showed that all bands detectable with the antibody were bound to heads and to midpieces and associated large tail fragments; no bands were detected in either small tail or membrane fragments or in the cytoplasmic fraction. Differential extraction of the midpiece/large tail fraction revealed two protein bands with closely similar electrophoretic mobilities to the 96- and 59-kDa phosphorylated bands; MALDI-TOF analyses of these bands revealed both to be members of the Odf2 family.

Can foods with added soya-protein or fish-oil reduce risk factors for coronary disease? A factorial randomised controlled trial.

BACKGROUND AND AIMS: To develop functional foods which are capable of reducing key risk factors for coronary heart disease in an at risk population. The specific hypothesis tested here was that providing bread, cracker biscuits and snack bars fortified with DHA (long-chain omega 3) and soya-protein would havd a positive impact on cholesterol and blood pressure. METHODS AND RESULTS: A pragmatic, double-blind, factorial placebo-controlled randomised trial recruiting 213 middle-aged men and women with untreated elevated total cholesterol or blood pressure. The factors examined were the effect of giving supplies of bread, cereal bars and cracker biscuits fortified with 2 g fish oils (DHA, 22: 6n-3), or 25 g soya-protein (containing 50 mg of isoflavonoids) for five weeks. Primary and secondary outcomes included total, low-density and high-density cholesterol (HDL-C), and systolic and diastolic blood pressure. Compliance was assessed using biomarkers and food intake histories. DHA enriched foods increased HDL-C by 6.0% (95% CI 2.5%, 9.6%) but had no effect on total or low-density cholesterol or blood pressure. Overall, soya-protein did not influence any of the outcomes assessed. However, in women only, soya-protein increased systolic blood pressure by 5.9% (95% CI 1.73, 9.9%). CONCLUSIONS: Adding DHA (fish-oils) to staple foods might supplement existing methods to help reduce CVD morbidity and mortality. However, these findings highlight the importance of ensuring that functional foods do not present any harms to particular subgroups within a general population, if they are to be made freely available to consumers. This latter point requires further attention by the research community in relation to soya-protein.

Role of potassium channels, the sodium-potassium pump and the cytoskeleton in the control of dog sperm volume.

Response to osmotic shock is an important aspect of mammalian sperm physiology. In this study we recorded volume changes of dog spermatozoa at 39, 33, and 25 degrees C under isotonic conditions and following hypotonic shock. Cell volume measurements were performed electronically in saline solutions of 300 and 150 mOsmol kg(-1), and Percoll-washed preparations were compared with unwashed samples. The involvement of potassium channels in volume control was tested by treatment with quinine, while the involvement of the plasma membrane Na(+)-K+ pump was tested by treatment with ouabain. The role of the cytoskeleton was investigated by treatment with colchicine and cytochalasin D. The number of cell populations observed varied with temperature and tonicity. In both types of sperm preparations, between two and three populations were present under isotonic conditions at 25 degrees C whereas at 39 and 33 degrees C only one population was detected. Hypotonic stress at the higher temperatures caused the single population to swell, whereas at 25 degrees C it resulted in a population of cells whose modal volume was similar to that of the middle isotonic sub-population. Both quinine and the cytoskeletal inhibitors markedly increased swelling both under hypotonic conditions at 39 degrees C and under isotonic conditions at 25 degrees C. However, little or no effect of ouabain was observed. We conclude that in dog spermatozoa swelling in response to hypotonic conditions is minimised through the activity of potassium channels and the presence of an intact cytoskeletal network. Under isotonic conditions at 25 degrees C, a considerable proportion of the sperm population is already swollen; this swelling varies between individual males and appears to be due to lowered cytoskeletal and potassium channel activity.

Novel sequences encoding venom C-type lectins are conserved in phylogenetically and geographically distinct Echis and Bitis viper species.

Envenoming by Echis saw scaled vipers and Bitis arietans puff adders is the leading cause of death and morbidity in Africa due to snake bite. Despite their medical importance, the composition and constituent functionality of venoms from these vipers remains poorly understood. Here, we report the cloning of cDNA sequences encoding seven clusters or isoforms of the haemostasis-disruptive C-type lectin (CTL) proteins from the venom glands of Echis ocellatus, E. pyramidum leakeyi, E. carinatus sochureki and B. arietans. All these CTL sequences encoded the cysteine scaffold that defines the carbohydrate-recognition domain of mammalian CTLs. All but one of the Echis and Bitis CTL sequences showed greater sequence similarity to the beta than alpha CTL subunits in venoms of related Asian and American vipers. Four of the new CTL clusters showed marked inter-cluster sequence conservation across all four viper species which were significantly different from that of previously published viper CTLs. The other three Echis and Bitis CTL clusters showed varying degrees of sequence similarity to published viper venom CTLs. Because viper venom CTLs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis and Bitis CTLs on the basis of sequence alone. The extraordinary level of inter-specific and inter-generic sequence conservation exhibited by the Echis and Bitis CTLs leads us to speculate that antibodies to representative molecules should neutralise the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East and the Indian subcontinent.

Molecular cloning of phospholipases A(2) from venom glands of Echis carpet vipers.

Venom toxin-specific antibodies offer a more rational treatment of snake envenoming than conventional antivenom. Here, we describe novel cDNAs encoding phospholipase A(2) (PLA(2)) isoforms from venom gland RNA of Echis pyramidum leakeyi (Epl), Echis sochureki (Es) and Echis ocellatus (Eo). The deduced amino acid sequences of these cDNAs encoded proteins with high overall sequence identity to the viper group II PLA(2) protein family, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA(2) enzymes. Comparison of the PLA(2) sequences from Echis with those from related vipers failed to make significant geographic, taxonomic or PLA(2)-function distinctions between these Echis PLA(2) isoforms. However, their deduced hydrophilicity profiles revealed a conserved tertiary structure that we will exploit, by epidermal DNA immunization, to generate PLA(2)-neutralizing antibodies with polyspecific potential.

Cyclic AMP signalling during mammalian sperm capacitation--still largely terra incognita.

Cyclic AMP is known to play a major role in intracellular signalling during mammalian sperm capacitation. However, despite much research, many of the molecular details of cyclic AMP's involvement remain obscure. In this review, I discuss the following aspects, presenting some original data as illustration where relevant. With respect to cyclic AMP synthesis, uncertainties exist as to the number of forms of adenylyl cyclase that are present in the spermatozoon, whether they are cytosolic or bound to subcellular structures, and which physiological effectors they respond to (e.g. bicarbonate, Ca2+, or receptor-coupled G-proteins). While net intracellular levels of cyclic AMP in spermatozoa depend upon the relative activities of adenylyl cyclase and phosphodiesterase, there are wide between-sample variations within species, both in basal levels and in levels attained after activation of the cyclase (e.g. after sperm treatment with bicarbonate). Moreover, minor changes in bulk cyclic AMP levels can result in large changes in cyclic AMP-dependent functions. Finally, while cyclic AMP levels respond very rapidly to sperm treatment by effectors such as bicarbonate and Ca2+ (key components of capacitating media), there are big discrepancies between the rates of functional response. For example, enhancement of motility and collapse of phospholipid asymmetry take place within a few minutes, whereas more than 1 h of exposure to capacitating conditions is needed for cyclic AMP-dependent protein tyrosine phosphorylation to become detectable or for the sperm population to attain a capacitated state.