Identification of Floral Volatiles and Pollinator Responses in Kiwifruit Cultivars, Actinidia chinensis var. chinensis.

Volatiles emitted from unpollinated in situ flowers were collected from two male cultivars, 'M33', 'M91', and one female cultivar 'Zesy002' (Gold3) of kiwifruit (Actinidia chinensis var. chinensis). The samples were found to contain 48 compounds across the three cultivars with terpenes and straight chain alkenes dominating the headspace. Electrophysiological responses of honey bees (Apis mellifera) and bumble bees (Bombus terrestris) to the headspace of the kiwifruit flowers were recorded. Honey bees consistently responded to 11 floral volatiles from Gold3 pistillate flowers while bumble bees consistently responded to only five compounds from the pistillate flowers. Nonanal, 2-phenylethanol, 4-oxoisophorone and (3E,6E)-α-farnesene from pistillate flowers elicited responses from both bee species. Overall, honey bees were more sensitive to the straight chain hydrocarbons of the kiwifruit flowers than the bumble bees, which represented one of the main differences between the responses of the two bee species. The floral volatiles from staminate flowers of the male cultivars 'M33' and 'M91' varied greatly from those of the pistillate flowers of the female cultivar Gold3, with most of the bee active compounds significantly different from those in the Gold3 flower headspace. The total floral emissions of 'M33' flowers were significantly less than those of the Gold3 flowers, while the total floral emissions of the 'M91' flowers were significantly greater than those of the Gold3 flowers.

A consortium of non-rhizobial endophytic microbes from Typha angustifolia functions as probiotic in rice and improves nitrogen metabolism.

Endophytic microbes isolated from plants growing in nutrient-deficient environments often possess properties that improve nutrition of agriculturally important plants. A consortium of non-rhizobial endophytic microbes isolated from a macrophyte Typha angustifolia growing in the marginal wetlands associated with a Uranium mine was characterized for their beneficial effect on rice and the mechanisms of growth promotion were investigated. The microbes were identified and characterized for their potential plant growth promoting (PGP) properties. Effect of these microbes on nitrogen (N)-metabolism of rice was tested as Typha endophytes were predominantly (N)-fixing. Relative N-use efficiency and expression of genes involved in N-uptake and assimilation were investigated in treated plants. Evidence of horizontal gene transfer (HGT) of dinitrogen reductase gene was observed within the consortium from a Pseudomonas stutzeri strain. The consortium behaved as plant probiotic and showed substantial growth benefits to Typha, their natural host as well as to rice. Typha endophytes colonized rice endosphere significantly increasing biomass, shoot length and chlorophyll content in rice plants both under N-sufficient and N-deficient conditions. N-uptake and assimilation genes were upregulated in plants treated with the endophytes even after three weeks post infection. Our results suggested, HGT of nitrogen-fixation trait to be highly prevalent among endophytes isolated from nutrient-poor habitats of the uranium mine. A long-term nitrogen deficiency response in the treated plants was elicited by the consortium improving N-uptake, assimilation and relative N-use efficiency of rice plants. This appeared to be at least one of the main strategies of plant growth promotion.

A pilot study of scoliosis assessment using radiation free surface topography in children with GMFCS IV and V cerebral palsy.

BACKGROUND: Prevalence of scoliosis in cerebral palsy (CP) parallels the extent of neurological impairment and causes significant morbidity. Monitoring is important but requires regular radiological investigation. Surface topography provides a non-radiological approach to scoliosis monitoring. AIM: To evaluate validity, reproducibility and feasibility of Quantec(®) scans to monitor scoliosis in children with severe CP. METHODS: Twenty non-ambulant children with CP, Gross Motor Function Classification System (GMFCS) grade IV/V had clinical, radiological and Quantec spinal assessment. The children were supported during scans using a seating system specifically designed for this study. Validity was assessed by comparing Quantec (Q) angle with gold standard (Cobb angle), reproducibility analysed using Bland-Altman plots and feasibility assessed using a questionnaire. RESULTS: Prevalence of scoliosis on radiological examination was 65%. Of these children, 85% had curves with Cobb angle less than 28°. Quantec scanning was feasible with appropriate postural support. Mean (and standard deviation) for differences between Cobb and Quantec (Q) angle were 0.02° (6.2°) and for Quantec inter-observer variability were 0.5° (5.8°). CONCLUSIONS: Quantec scanning was feasible, reproducible and had good validity when compared with Cobb angle in a supportive seating system. To consolidate these findings a further study needs to be undertaken with larger number of children with Cobb angles between 25° and 45°.

Targeted yield concept and a framework of fertilizer recommendation in irrigated rice domains of subtropical India.

Soil test crop response (STCR) correlation studies were carried out in Vindhyan alluvial plain during 2001 to 2004 taking IR-36 as test crop to quantify rice production in the context of the variability of soil properties and use of balanced fertilizers based on targeted yield concept. The soils were developed on gently sloping alluvial plain with different physiographic settings and notable variation in drainage condition. Soil properties show moderate variation in texture (loamy to clay), organic carbon content (4.4 to 9.8 g/kg), cation exchange capacity (10.2 to 22.4 cmol (p+)/kg) and pH (5.3 to 6.4). Soil fertility status for N is low to medium (224 to 348 kg/ha), P is medium to high (87 to 320 kg/ha) and K ranges from medium to high (158 to 678 kg/ha). Database regarding nutrient requirement in kg/t of grain produce (NR), the percent contribution from the soil available nutrients [CS (%)] and the percent contribution from the applied fertilizer nutrients [CF (%)] were computed for calibrating and formulating fertilizer recommendations. Validity of the yield target for 7 and 8 t/ha was tested in farmers' fields and yields targets varied at less than 10%. The percent achievement of targets aimed at different level was more than 90%, indicating soil test based fertilizer recommendation approach was economically viable within the agro-ecological zone with relatively uniform cropping practices and socio-economic conditions.

Subdural haematoma and effusion in infancy: an epidemiological study.

AIM: To determine incidence, aetiology, and clinical features of subdural haematoma and effusion (SDH/E) in infancy throughout the British Isles. METHODS: Cases were notified to the British Paediatric Surveillance Unit over 12 months by paediatricians, neurosurgeons, and paediatric and forensic pathologists. RESULTS: A total of 186 infants (121 boys, 65 girls) aged 0-2 years were identified. Annual incidence of SDH/E for the UK and Republic of Ireland is 12.54/100,000 aged 0-2 (95% CI 10.3 to 14.62) and 24.1/100,000 aged 0-1 (95% CI 20.89 to 28.18). A total of 106 infants suffered non-accidental head injury (NAHI), 7 accidental head injury, 26 a perinatal cause, 7 a non-traumatic medical condition, 23 meningitis, and in 17 the cause was undetermined; 35 infants died. Significant differences were found in injury pattern, body weight, and Townsend score between NAHI and SDH/E from other cause. There were fewer diagnostic investigations in non-NAHI cases. Delay in diagnosis of greater than a week occurred in 48/181. CONCLUSION: SDH/E is a significant cause of morbidity and mortality in infancy. NAHI is the predominant cause of SDH/E. SDH/E can present in a non-specific and varied way and must be considered in any infant who is unwell. Determining the cause of the SDH/E in some cases continues to present a diagnostic challenge.

Glucose metabolism in traumatic brain injury: a combined microdialysis and [18F]-2-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) study.

Following traumatic brain injury, as a consequence of ionic disturbances and neurochemical cascades, glucose metabolism is affected. [18F]-2-Fluoro-2-deoxy-D-glucose (FDG) Positron Emission Tomography (FDG-PET) provides a measure of global and regional cerebral metabolic rate of glucose (rCMRglc), but only during the time of the scan. Microdialysis monitors energy metabolites over extended time periods, but only in a small focal volume of the brain. Our objective in this study is to assess the association of parameters derived from these techniques when applied to patients with traumatic brain injury. Eleven sedated, ventilated patients receiving intracranial pressure monitoring and managed using Addenbrooke's Neurosciences Critical Care Unit protocols were monitored. Dialysate values for glucose, lactate, pyruvate, and glutamate, and the lactate to glucose (L/G), lactate to pyruvate (L/P) and pyruvate to glucose (P/G) ratios were determined and correlated with rCMRglc. FDG-PET scans were performed within 24 hours (five patients), or between 1 and 4 days (two patients) or after 4 days (six patients). Two patients were rescanned 4 and 7 days after their initial scan. A 20 mm region of interest (ROI) was defined on co-registered CT scan on two contiguous slices around the microdialysis catheter. Mean (+/-sd) for rCMRglc was 19.1 +/- 5.5 micromol/100 g/min, and the corresponding microdialysis values were: glucose 1.4 +/- 1.4 mmol/ L; lactate 5.3 +/- 3.6 mmol/L; pyruvate 164.1 +/- 142.3 micromol/L; glutamate 15.0 +/- 14.7 micromol/L; L/G 11.0 +/- 16.0; L/P 27.3 +/- 7.9 and P/G 381 +/- 660. There were significant relations between rCMRglc and dialysate lactate (r = 0.58, P = 0.04); pyruvate (r = 0.57, P = 0.04), L/G (r = 0.55, P = 0.05), and the P/G (r = 0.56, P = 0.05) but not between rCMRglc and dialysate glucose, L/P or glutamate in this data set. The results suggest that increases in glucose utilization as assessed by FDG-PET in these patients albeit in mainly healthy tissue are associated with increases in dialysate lactate, pyruvate, L/G and the P/G ratio perhaps indicating a general rise in metabolism rather than a shift towards non-oxidative metabolism. Further observations are required with regions of interest (microdialysis catheters positioned) adjacent to mass lesions notably contusions.

Review of policies and guidelines on infant feeding in emergencies: common ground and gaps.

Recent crises in regions where exclusive breastfeeding is not the norm have highlighted the importance of effective policies and guidelines on infant feeding in emergencies. In 1993, UNICEF compiled a collection of policy and guideline documents relating to the feeding of infants in emergency situations. In June 2000 Save the Children, UK, UNICEF and the Institute of Child Health undertook a review of those documents, updating the list and identifying the common ground that exists among the different policies. The review also analysed the consistency of the policy framework, and highlighted important areas where guidelines are missing or unclear. This article is an attempt to share more widely the main issues arising from this review. The key conclusions were that, in general, there is consensus on what constitutes best practice in infant feeding, however, the lack of clarity in the respective responsibilities of key UN agencies (in particular UNICEF, UNHCR and WFP) over issues relating to co-ordination of activities which affect infant-feeding interventions constrains the implementation of systems to support best practice. Furthermore, the weak evidence base on effective and appropriate intervention strategies for supporting optimal infant feeding in emergencies means that there is poor understanding of the practical tasks needed to support mothers and minimise infant morbidity and mortality. We, therefore, have two key recommendations: first that the operational UN agencies, primarily UNICEF, examine the options for improving co-ordination on a range of activities to uphold best practice of infant feeding in emergencies; second, that urgent attention be given to developing and supporting operational research on the promotion of optimal infant-feeding interventions.

From policy to practice: challenges in infant feeding in emergencies during the Balkan crisis.

The preparation and dissemination of policy statements are necessary but insufficient to prevent the inappropriate use of infant-feeding products in emergencies. The widespread failure of humanitarian agencies operating in the Balkan crisis to act in accordance with international policies and recommendations provides a recent example of the failure to translate infant-feeding policies into practice. This article explores the underlying reasons behind the failures which include: (1) the weak institutionalisation of policies; (2) the massive quantities of unsolicited donations of infant-feeding products: (3) the absence of monitoring systems; (4) inadequate co-ordination mechanisms; (5) the high costs of correcting mistakes; and (6) the cumulative effects of poor practice. Efforts to uphold best practice during the crisis are also documented. Finally, the article identifies actions that could be undertaken in advance of and during future emergencies to enhance the application of infant feeding policies in emergencies.

Production of safe therapeutic feeds from contaminated water supplies.

We have assessed the effectiveness of using an osmotic sachet for safe rehydration of therapeutic milk from contaminated water supplies in a poor urban area of Bangladesh. 35 women were given sachets to hydrate in their homes and comparison of the hydration water and the reconstituted milk showed that the high bacterial contamination reported in the domestic water supply was removed by use of the sachet. The mean rehydration time was 4.5 h (range 3.4-5.5). This rehydration method could have a role in the preparation of therapeutic feeds where microbiological contamination of the environment is a serious problem and availability of adequate water is not a limiting factor.

Poor nutrition is a serious problem in children with cerebral palsy in Palawan, the Philippines.

Children with cerebral palsy (CP) in developed countries have poor nutritional status; however there is little data from developing countries. In Palawan, in the Philippines, the nutritional status of 31 children with CP was compared to that of their siblings (n = 20) and a control group of neighbourhood children (n = 64), matched for age and sex. The children's weights, heights and armspans were measured. The heights of children with CP could not be measured and were estimated from their armspans using an equation relating height to armspan in siblings and controls. Haemoglobin levels of the study cases and siblings were measured. Siblings and controls had similar nutritional status. The children with CP had extremely poor nutritional status, and had significantly smaller weights for height, heights for age and weights for age than siblings or controls. Haemoglobin levels were not significantly different between the children with CP and their siblings. The nutritional status of children with quadriplegic CP was much poorer than that of similar children in the USA. The severity of malnutrition in children with CP is likely to be detrimental to their development, and a nutritional component should be incorporated into rehabilitation programmes. Also, there is a need to examine the nutritional status of children with CP in other developing countries.

Adolescent nutrition in a rural community in Bangladesh.

The objective of this study was to assess the nutritional status of adolescent boys and girls in a rural community in Bangladesh. Between December 1996 and January 1997, a cross-sectional survey was carried out in 803 households, each containing at least one adolescent, sampled consecutively from four purposely-selected villages in Rupganj Thana, Narayanganj district. Initially, the guardians of 1483 healthy and unmarried 10-17 year old adolescents (51% boys and 49% girls) were interviewed about family structure and socio-economic status. Out of these children, 906 (47% boys and 53% girls) from 597 households were weighed, had their height and MUAC measured and were clinically examined. Blood was then collected from 861 adolescents for haemoglobin estimation. The median monthly income per person in these 597 families was approximately Taka 554 (US $12). Twenty seven per cent of the household heads were labourers, 21% were solvent farmers, 14% ran small scale businesses and 6% were unemployed. Sixty seven per cent of adolescents were thin (defined as BMI < 5th centile of WHO recommended reference) with 75% boys and 59% girls being affected. The percentage of thin adolescents fell from 95% at age 10 years to 12% at age 17 years. The prevalence of stunting (height for age < 3rd centile NCHS/WHO) was 48% for both boys and girls and rose from 34% at age 10 to 65% at age 17. On clinical examination angular stomatitis was present in 46%, 27% had glossitis, 38% had pallor, 11% had dental caries, 3.2% had an conspicuously enlarged thyroid and 2.1% had eye changes of vitamin A deficiency. According to INACG (International Nutritional Anaemia Consultative Group, 1985) cut-off values, 94% of the boys and 98% of the girls were anaemic. We conclude that rural Bangladesh adolescents suffer from high rates of malnutrition and almost universal anaemia. Nutritional interventions to target this population are urgently required.

Nutritional status and age at menarche in a rural area of Bangladesh.

The age at menarche and its association with nutritional status in a rural area of Bangladesh was determined. A cross-sectional study was conducted in four villages of Rupganj Thana of Narayanganj district. Data was collected through October to December 1996 using a pre-tested structured questionnaire interview schedule, and nutritional status was measured by weight, height, body mass index (BMI) and physical examination. Data were obtained on 436 adolescent girls aged 10-17 years. Among them, 165 (37.8%) girls had commenced menarche. The mean age at menarche as determined by retrospective recall was 13 years SD 0.89 (n = 165). The median age at menarche determined by the status quo method was 13.0. Among the adolescents 60.1% were thin (BMI < 5th centile WHO recommended reference) and 48.2% were stunted (< 3rd centile NCHS/WHO). The mean weight and BMI were significantly higher among the menstruating girls of 13, 14 and 15 years (p < 0.01) than non-menstruating girls. The mean height was found to be significantly higher at 11-14 years among the menstruating girls (p < 0.05). A lower prevalence of angular stomatitis was found among the menstruating adolescent girls compared with the non-menstruating girls, 36.4% versus 46.5%, although this was statistically non-significant (odds ratio = 0.66, 95% CI 0.43-1.00). For glossitis, no significant difference was found. Among the menstruating girls 12.1% were suffering from menorrhagia and 31.5% from dysmenorrhoea. We conclude that the age of menarche among this rural Bangladeshi community is not as delayed as expected. Not surprisingly, menarche is associated with better nutritional status. The surveyed population had extremely high rates of undernutrition which suggests that adolescents in this and similar situations require specific intervention programmes to improve their nutritional status.

Autophosphorylation-dependent activation of a calcium-dependent protein kinase from groundnut

Ca2+-dependent protein kinases (CDPKs) containing a calmodulin-like domain integrated in their primary sequence are present primarily in plants. A member of this family was characterized from the groundnut (Arachis hypogea) plant and called GnCDPK (M. DasGupta [1994] Plant Physiol 104: 961-969). GnCDPK specifically uses the myosin light chain synthetic peptide (MLCpep), which is the phosphate-accepting domain of smooth muscle myosin light chains (KKRPQRATSNVFS), as an exogenous substrate under in vitro experimental conditions. In this report we show that GnCDPK undergoes intramolecular autophosphorylation. This self-phosphorylation occurs in threonine residues in a Ca2+-dependent (K0.5 = 0.5 &mgr;M) and calmodulin-independent manner. The kinase activity toward MLCpep and its sensitivity to Ca2+ were unaffected by prior autophosphorylation when measured under saturating ATP concentrations. The role of autophosphorylation in the exogenous substrate MLCpep phosphorylation reaction was reinvestigated at low ATP concentrations. A pronounced lag time of 1 to 2 min, followed by a linear increase of activity for 7.5 min, was seen in the initial rate of MLCpep phosphorylation under such suboptimal conditions. Prior autophosphorylation completely abolished this lag phase, and a sharp rise of exogenous substrate phosphorylation was seen from the 1st min. Our results suggest that autophosphorylation is a prerequisite for the activation of GnCDPK.

Predictors of outcome after radiofrequency catheter ablation of the atrioventricular node for atrial fibrillation and congestive heart failure.

BACKGROUND: Although radiofrequency catheter ablation of the atrioventricular (AV) node is an established treatment for atrial fibrillation (AF) with uncontrolled ventricular response, factors that predict clinical outcome in patients with associated congestive heart failure (CHF) are unknown. METHODS AND RESULTS: AV node ablation and permanent pacemaker implantation was performed in 44 consecutive patients (mean age 71+/-10 years) with CHF and AF associated with uncontrolled ventricular response. Immediately before ablation, mean left ventricular ejection fraction (EF) measured by 2-dimensional echocardiogram was 34.6%+/-9.8%, mean exercise tolerance time was 2.6+/-1.8 minutes, and mean quality of life score was 62.3+/-19.7. Complete AV block was achieved in all 44 patients but was complicated by death in 1 patient from cardiogenic shock soon after ablation. By 1 month after ablation, EF increased to 43.8%+/-13.7% (P < .01), exercise tolerance time was 4.0+/-2.5 minutes (P < .01), and mean quality of life score decreased to 35.6+/-18.1 (P < .01). Improved cardiac performance (increase in EF > or = 9% over baseline EF) was detected in 20 (45%) of the patients. During a mean follow-up of 17+/-9 months, 5 patients died suddenly of presumed ventricular tachyarrhythmia and 4 others died of progressive CHF. Multivariate Cox survival analysis identified baseline EF < or = 30%, presence of significant mitral regurgitation (>2+) before ablation, and failure to exhibit improved cardiac performance by 1 month after ablation as the only independent predictors of death. CONCLUSIONS: Baseline variables and failure of EF to improve soon after AV node ablation identifies patients with CHF and AF who have a high mortality rate. Adjunctive therapy to reduce sudden death and progressive heart failure should be evaluated in this subgroup.

Comparison of the effects of AV nodal ablation versus AV nodal modification in patients with congestive heart failure and uncontrolled atrial fibrillation.

Radiofrequency (RF) catheter ablation of the atrioventricular node (AVN) and implantation of a ventricular pacemaker can improve cardiac performance in patients with congestive heart failure (CHF) and uncontrolled atrial fibrillation (AF). Alternatively, RF catheter modification of the AVN has been proposed to slow ventricular response during AF without requirement for permanent pacing. Among 44 consecutive patients (mean age 69.7 +/- 10.2 years) with drug resistant chronic AF, 22 (group I) had AVN ablation with permanent ventricular pacemaker implantation, while 22 patients had attempted AVN modification. Complete AV block was obtained in all group I patients while only seven (32%) AVN modification patients (group II) had permanent slowing of ventricular rate. Among patients in group I, mean left ventricular ejection fraction (EF) increased from 32.2% +/- 8.8% before ablation to 41.9% +/- 14.6% 4-weeks postablation (P < 0.01); exercise tolerance time (ETT) increased from 2.9 +/- 2.2 minutes to 4.5 +/- 2.9 minutes (P < 0.01); and quality-of-life score decreased from 66.1 +/- 22.6 to 36.9 +/- 17.1 (P < 0.01). By comparison, there was only a small increase in ETT in the seven successful group II patients (2.4 +/- 1.8 minutes to 3.0 +/- 1.9 minutes; P < 0.05) and there was no significant change in EF or quality-of-life. While AVN ablation can occasionally have transient adverse effects, it is more effective than AVN modification for improving cardiac performance in selected patients with CHF and AF.

Optimism, social support, and premenstrual dysphoria.

The relationships between dispositional optimism, social support, and mood and performance symptoms associated with the premenstrum were examined in a sample of 101 adult women. Optimism and satisfaction with one's level of social support were positively correlated. Optimism was related inversely to mood-related symptoms, even after controlling for the effects of social support. Optimism did not correlate with premenstrual performance impairment. Social support did not correlate with any premenstrual symptoms.

Young women's sexual risk taking behaviour: re-visiting the influences of sexual self-efficacy and sexual self-esteem.

This study investigates the influences on young women's sexual risk taking of (a) attitudes such as sexual self-efficacy and sexual self-esteem and (b) personal and sexual characteristics, such as age, the age of first sexual experience, the number of sexual partners and reported overall amount of sex. Three hundred and thirty-one young women participated in this study. With respect to regular sexual relationships, the results show that risk taking behaviour is directly and positively associated with sexual self-esteem. The effect of sexual self-efficacy on risk taking in regular relationships, however, is indirect and positive, being mediated by the overall amount of sexual activity. In contrast, with respect to casual sexual relationships, risk taking behaviour is negatively associated with self-esteem and positively associated with sexual self-efficacy. In addition, sexual self-esteem influences casual risk taking indirectly and positively, the effect being mediated by the overall amount of sexual activity. The findings in relation to sexual self-esteem and sexual self-efficacy are contrary to the expectations that these variables would be negatively associated with sexual risk taking behaviours. The implications for safe sex education are discussed.

Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells.

1. The actions of the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists, memantine (1-amino-3,5-dimethyladamantane) and (+)-MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, dizocilpine), on recombinant NMDA receptors has been studied by use of the whole-cell patch clamp technique. 2. Human embryonic kidney (HEK) 293 cells were transiently transfected with different NMDA receptor subunit combinations (NR1a/NR2A, NR1a/NR2B and NR1a/NR2D). A mutant form of the green fluorescent protein (GFP) cotransfected with the NMDA receptor subunits to enable the visualization of transfected cells. 3. Memantine (0.3-30 microM) blocked L-glutamate (100 microM)-mediated currents in a concentration-dependent manner in NR1a/NR2A, NR1a/NR2B and NR1a/NR2D transfected cells with IC50 values (at -70 mV) of 0.93 +/- 0.15 microM, 0.82 +/- 0.12 microM and 0.47 +/- 0.06 microM (mean +/- s.c. mean), respectively. 4. The memantine-induced block was strongly voltage-dependent. Alteration of the holding potential from -70 mV to +60 mV resulted in an e-fold increase in the IC50 values per 30-33 mV change in membrane potential, for all 3 subunit combinations investigated. 5. The kinetics of the actions of memantine (30 microM) were investigated for the NR1a/2A combination, in 6 cells (13-15 determinations). At -70 mV, the block and recovery from block were both best described by two exponentials with time-constants of 201 +/- 23 ms (81 +/- 2%) and 3.9 +/- 0.6 s and 597 +/- 94 ms (18 +/- 1%) and 18.6 +/- 2.4 s, respectively. The predominant effect of depolarization was to increase the weight of the faster recovery time-constant. Kinetic analysis suggests that these results are consistent with previously proposed Markov models. 6. (+)-MK-801 was studied briefly for comparative purposes. (+)-MK-801 (200 nM) preferentially blocked NMDA receptor currents (at -70 mV) in NR1a/NR2A and NR1a/NR2B (82 +/- 10% and 93 +/- 2% depressions) compared to NR1a/NR2D (38 +/- 7%) transfected cells. (+)-MK-801 appeared to be less voltage-dependent than memantine on all three receptor combinations. 7. In conclusion, memantine was a voltage-dependent antagonist of recombinant rat NMDA receptors expressed in HEK 293 cells but showed little selectivity between the subunits investigated. Its actions on these recombinant receptor combinations are similar to its actions on native NMDA receptors.

An investigation of the membrane topology of the ionotropic glutamate receptor subunit GluR1 in a cell-free system.

We have utilized cell-free translation in rabbit-reticulocyte lysate supplemented with canine pancreatic microsomal membranes to study the processing and membrane topology of the rat ionotropic glutamate receptor subunit GluR1. In vitro-synthesized RNA encoding GluR1 was translated to yield a primary translation product with an apparent molecular mass of 99 kDa. In the presence of microsomal membranes this protein was processed to give a band of 107 kDa. Treatment with peptide-N-glycosidase F showed that this increase in molecular mass was due to N-linked glycosylation. Incubation of the processed receptor with proteinase K revealed the presence of a 68 kDa protease-resistant band which decreased to 56 kDa following deglycosylation. A deletion mutant (GluR1M1) lacking the predicted transmembrane domains was fully translocated across the microsomal membrane and protected from the action of the protease. The mutant and wild-type receptor could be immunoprecipitated by anti-peptide antibodies directed against the C-terminus. Following translocation of the wild-type and mutant receptor across the microsomal membrane and treatment with proteinase K the antibody binding to GluR1 was abolished, but was retained for GluR1M1. These data allow identification of the orientation of the N- and C-termini of GluR1 within the microsome; results which are consistent with an extracellular N-terminal and intracellular C-terminal localization following incorporation into the plasma membrane.