Venezuelan equine encephalitis virus glycoprotein pseudotyping confers neurotropism to lentiviral vectors.

We have produced high-titre HIV-1 green fluorescent protein-expressing lentiviral (LV) vectors pseudotyped with strain 3908 Venezuelan equine encephalitis virus glycoprotein (VEEV-G) and used them to study transduction of: (1) rat embryonic motor neuron (MN) and striatal neuron primary cultures, (2) differentiated MN cell line NSC-34 and (3) adult rat striatum. In primary neuronal cultures, transduction with VEEV-G-pseudotyped LV was more efficient and more neuronal than with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped LV. In NSC-34 cells clear retrograde transport of VEEV-G vector particles was observed. In the striatum at the injection site, transduction with the VEEV-G vectors driven by cytomegalovirus or phosphoglycerate kinase promoters exhibited a distinct neuronal tropism with no microglial and only a minor astroglial component, superior to that obtained with VSV-G-pseudotyped LV, irrespective of the promoter used. Neuronal transduction efficiency increased over time. Distal to the injection site transduction of mitral cells in the olfactory bulb, thalamic neurons and dopaminergic neurons in the substantia nigra pars compacta was detected. This, together with observations of retrograde axonal trafficking in vitro indicates that these vectors also possess low level of retrograde neuronal transduction capability in vivo. In this study, we demonstrate both strong neurotropism as well as sustainability of expression and minimal host immune response in vivo, making the VEEV-G-pseudotyped LV vectors potentially useful for gene therapy of neurodegenerative diseases.

Clinical effectiveness and cost-effectiveness of imatinib dose escalation for the treatment of unresectable and/or metastatic gastrointestinal stromal tumours that have progressed on treatment at a dose of 400 mg/day: a systematic review and economic evaluation.

BACKGROUND: Imatinib dose escalation is advocated for gastrointestinal stromal tumour (GIST) treatment, but its effectiveness compared with sunitinib and best supportive care (BSC) after failure at the 400 mg/day dose is unknown. OBJECTIVES: To assess the effectiveness and cost-effectiveness of imatinib at escalated doses of 600 or 800 mg/day for patients with unresectable and/or metastatic GISTs whose disease had progressed on 400 mg/day. DATA SOURCES: Electronic databases, including MEDLlNE, MEDLINE In-Process, EMBASE, BIOSIS, Science Citation Index, Health Management Information Consortium and the Cochrane Controlled Trials Register, were searched until September 2009. REVIEW METHODS: A systematic review of the literature was carried out according to standard methods. An economic model was constructed to assess the cost-effectiveness of seven alternative pathways for treating patients with unresectable and/or metastatic GISTs. RESULTS: Five primary studies involving 669 people were included for clinical effectiveness; four reported imatinib and one reported sunitinib. The data were essentially observational as none of the studies was designed to specifically assess treatment of patients whose disease had progressed on 400 mg/day imatinib. For 600 mg/day imatinib, between 26% and 42% of patients showed either a partial response (PR) or stable disease (SD). Median time to progression was 1.7 months (range 0.7-24.9 months). For 800 mg/day imatinib, between 29% and 33% of patients showed either a PR or SD. Median overall survival (OS) was 19 months [95% confidence interval (CI) 13 to 23 months]. Progression-free survival ranged from 81 days to 5 months (95% CI 2 to 10 months). Median duration of response was 153 days (range 37-574 days). Treatment progression led to 88% discontinuations but between 16% and 31% of patients required a dose reduction, and 23% required a dose delay. There was a statistically significant increase in the severity of fatigue (p < 0.001) and anaemia (p = 0.015) following dose escalation. For sunitinib, median OS was 90 weeks (95% CI 73 to 106 weeks). For the cost-effectiveness review, only one full-text study and one abstract were identified, comparing imatinib at an escalated dose, sunitinib and BSC, although neither was based on a UK context. The definition of BSC was not consistent across the studies, and the pattern of resources (including drugs for treatment) and measures of effectiveness also varied. Within the model, BSC (assumed to include continuing medication to prevent tumour flare) was the least costly and least effective. It would be the care pathway most likely to be cost-effective when the cost per quality-adjusted life-year threshold was < £25,000. Imatinib at 600 mg/day was most likely to be cost-effective at a threshold between £25,000 and £45,000. Imatinib at 600 mg/day followed by further escalation followed by sunitinib was most likely to be cost-effective at a threshold > £45,000. LIMITATIONS: The evidence base was sparse, data were non-randomised and potentially biased. The economic model results are surrounded by a considerable degree of uncertainty and open to biases of unknown magnitude and direction. CONCLUSIONS: Around one-third of patients with unresectable and/or metastatic GIST, who fail on 400 mg/day of imatinib, may show response or SD with escalated doses. Between a threshold of £25,000 and £45,000, provision of an escalated dose of imatinib would be most likely to be cost-effective. However, these results should be interpreted with caution owing to the limited evidence available on outcomes following imatinib dose escalation or sunitinib for this group of patients. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Systematic review and economic modelling of the effectiveness and cost-effectiveness of non-surgical treatments for women with stress urinary incontinence.

OBJECTIVES: To assess the clinical effectiveness and cost-effectiveness of non-surgical treatments for women with stress urinary incontinence (SUI) through systematic review and economic modelling. DATA SOURCES: The Cochrane Incontinence Group Specialised Register, electronic databases and the websites of relevant professional organisations and manufacturers, and the following databases: CINAHL, EMBASE, BIOSIS, Science Citation Index and Social Science Citation Index, Current Controlled Trials, ClinicalTrials.gov and the UKCRN Portfolio Database. STUDY SELECTION: The study comprised three distinct elements. (1) A survey of 188 women with SUI to identify outcomes of importance to them (activities of daily living; sex, hygiene and lifestyle issues; emotional health; and the availability of services). (2) A systematic review and meta-analysis of non-surgical treatments for SUI to find out which are most effective by comparing results of trials (direct pairwise comparisons) and by modelling results (mixed-treatment comparisons - MTCs). A total of 88 randomised controlled trials (RCTs) and quasi-RCTs reporting data from 9721 women were identified, considering five generic interventions [pelvic floor muscle training (PFMT), electrical stimulation (ES), vaginal cones (VCs), bladder training (BT) and serotonin-noradrenaline reuptake inhibitor (SNRI) medications], in many variations and combinations. Data were available for 37 interventions and 68 treatment comparisons by direct pairwise assessment. Mixed-treatment comparison models compared 14 interventions, using data from 55 trials (6608 women). (3) Economic modelling, using a Markov model, to find out which combinations of treatments (treatment pathways) are most cost-effective for SUI. DATA EXTRACTION: Titles and abstracts identified were assessed by one reviewer and full-text copies of all potentially relevant reports independently assessed by two reviewers. Any disagreements were resolved by consensus or arbitration by a third person. RESULTS: Direct pairwise comparison and MTC analysis showed that the treatments were more effective than no treatment. Delivering PFMT in a more intense fashion, either through extra sessions or with biofeedback (BF), appeared to be the most effective treatment [PFMT extra sessions vs no treatment (NT) odds ratio (OR) 10.7, 95% credible interval (CrI) 5.03 to 26.2; PFMT + BF vs NT OR 12.3, 95% CrI 5.35 to 32.7]. Only when success was measured in terms of improvement was there evidence that basic PFMT was better than no treatment (PFMT basic vs NT OR 4.47, 95% CrI 2.03 to 11.9). Analysis of cost-effectiveness showed that for cure rates, the strategy using lifestyle changes and PFMT with extra sessions followed by tension-free vaginal tape (TVT) (lifestyle advice-PFMT extra sessions-TVT) had a probability of greater than 70% of being considered cost-effective for all threshold values for willingness to pay for a QALY up to 50,000 pounds. For improvement rates, lifestyle advice-PFMT extra sessions-TVT had a probability of greater than 50% of being considered cost-effective when society's willingness to pay for an additional QALY was more than 10,000 pounds. The results were most sensitive to changes in the long-term performance of PFMT and also in the relative effectiveness of basic PFMT and PFMT with extra sessions. LIMITATIONS: Although a large number of studies were identified, few data were available for most comparisons and long-term data were sparse. Challenges for evidence synthesis were the lack of consensus on the most appropriate method for assessing incontinence and intervention protocols that were complex and varied considerably across studies. CONCLUSIONS: More intensive forms of PFMT appear worthwhile, but further research is required to define an optimal form of more intensive therapy that is feasible and efficient for the NHS to provide, along with further definitive evidence from large, well-designed studies.

Systematic review of the clinical effectiveness and cost-effectiveness of rapid point-of-care tests for the detection of genital chlamydia infection in women and men.

OBJECTIVE: To assess whether or not the Chlamydia Rapid Test (CRT) could improve detection of genital chlamydia, and whether it is more effective than current practice using nucleic acid amplification tests (NAATs), in terms of the number of cases of chlamydia that are detected and treated and the proportion of partners identified and treated. DATA SOURCES: Eleven electronic bibliographic databases (including MEDLINE and EMBASE) were searched until November 2008, as well as relevant websites. REVIEW METHODS: Studies of sexually active adolescent and adult women and men suspected of having or being tested for genital chlamydia infection were considered. The tests considered were the CRT and other comparator point-of-care tests identified, using a NAAT as a reference standard. Summary sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratios for each model were reported as a median and a 95% confidence interval (CI). Effectiveness was measured in terms of the absolute numbers of true-positives, false-positives, false-negatives (and other positive cases missed) and true-negatives detected. Costs were considered from the health service's perspective. Incremental cost-effectiveness ratios were used to examine the relative cost-effectiveness, and values of the major parameters of the models were varied in a sensitivity analysis. RESULTS: Thirteen studies enrolling 8817 participants were included in the analysis. In the pooled estimates for the CRT, sensitivity (95% CI) was 80% (73% to 85%) for vaginal swab specimens and 77% (59% to 89%) for first void urine (FVU) specimens. Specificity was 99% (99% to 100%) for vaginal swab specimens and 99% (98% to 99%) for FVU specimens. In the pooled estimates for a comparator point-of-care test (Clearview Chlamydia), sensitivity (95% CI) was 52% (39% to 65%) for vaginal, cervical and urethral swab specimens combined, and 64% (47% to 77%) for cervical specimens alone. Specificity was 97% (94% to 100%) for vaginal, cervical and urethral swab specimens combined, and 97% (88% to 99%) for cervical specimens alone. The results of the economic evaluation showed that for a hypothetical cohort of 1000 people, using the current practice of polymerase chain reaction testing would result in 12.63 people who were offered testing being correctly treated and having their sexual partners contacted, at a cost of 7070 pounds (for the whole cohort). For the CRT, the number being correctly treated would be 10.98, at a cost of 7180 pounds. For the Clearview Chlamydia test, the number correctly treated would be 7.14, at a cost of 7170 pounds. Both point-of-care tests were therefore more costly and less effective than current practice. CONCLUSIONS: The limited evidence available suggests that NAATs are still the most accurate and cost-effective method for diagnosing chlamydia infection. There may be circumstances in which point-of-care tests could be provided in addition to existing NAAT services, but there is currently little evidence on point-of-care methods in such settings. Robust evidence of the diagnostic accuracy of point-of-care tests for different types of samples is also still required, as are studies evaluating clinical effectiveness outcomes for these tests in comparison with NAATs.

Objective measurement of physical activity and sedentary behaviour: review with new data.

Objective methods are being used increasingly for the quantification of the amount of physical activity, intensity of physical activity and amount of sedentary behaviour in children. The accelerometer is currently the objective method of choice. In this review we address the advantages of objective measurement compared with more traditional subjective methods, notably the avoidance of bias, greater confidence in the amount of activity and sedentary behaviour measured, and improved ability to relate variation in physical activity and sedentary behaviour to variation in health outcomes. We also consider unresolved practical issues in paediatric accelerometry by critically reviewing the existing evidence and by providing new evidence.

Investigating couples' sleep: an evaluation of actigraphic analysis techniques.

'Blip' analysis, fast wavelet transformations (FWT) and correlation analysis have all been used to actigraphically assess the impact one person is having on another's sleep, yet no review exists as to the differences between, and applicability of, these methods for investigating couples' sleep. Using actigraphy data and audio sleep diaries collected from 18 couples, this paper provides such a review. This paper constructs and assesses two novel, analytical methods: Lotjonen's sleep/wake algorithm, and the partner impact on sleep wake analysis (PISWA). Both 'blip' analysis and correlation suggest that the strongest relationship between bed partners occurs on an epoch-to-epoch basis. However, 'blips' deal strictly with onset of movement and fail to incorporate strength and duration of movement. Conversely, correlation analysis incorporates some elements of strength and duration of movement but makes identification of onset problematic. FWT offer useful 'relativistic' pattern recognition, identifying onset, strength and duration of movement, but are difficult to quantify. Although audio diary data support the potential of Lotjonen's sleep/wake algorithm to identify sleep non-movement, sleep movement, wake non-movement (or quiet wakefulness) and wake movement, the problem remains that this method also relies on visualization. Of most promise, we argue, is the PISWA, which examines 'impact' of bed partners through incorporating elements of 'blip' analysis and the sleep/wake algorithm.

Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus.

Gonadotrophin-releasing hormone receptors (GnRH-Rs) are found in cancers of reproductive tissues, including those of the prostate, and gonadotrophin-releasing hormone (GnRH) can inhibit growth of cell lines derived from such cancers. Although pituitary and extra-pituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extra-pituitary GnRH-Rs have low affinity for GnRH analogues and may not activate phospholipase C or discriminate between agonists and antagonists in the same way as do pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in prostate cancer cells differ functionally from those of gonadotrophs. We found no evidence for endogenous GnRH-Rs in PC3 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at 10 plaque forming units (p.f.u.)/cell or greater, at least 80% of the cells expressed GnRH-Rs. These sites had high affinity (K(d )for [(125)I]Buserelin 1.1+/-0.4 nM) and specificity (rank order of potency: Buserelin> GnRH>>chicken (c) GnRH-II), and mediated stimulation of [(3)H]inositol phosphate (IP) accumulation. Increasing viral titre from 3 to 300 p.f.u./cell increased receptor number (2000 to 275 000 sites/cell respectively) and [(3)H]IP responses. GnRH also caused a biphasic increase in the cytoplasmic Ca(2+) concentration in Ad GnRH-R-infected cells but not in control cells. Mobilization of Ca(2+) from intracellular stores contributed to the spike phase of this response whereas the plateau phase was dependent upon Ca(2+) entry across the plasma membrane. This effect on Ca(2+) and stimulation of [(3)H]IP accumulation were both blocked by the GnRH-R antagonist, Cetrorelix. In addition, GnRH reduced cell number (as measured in MTT activity assays) and DNA synthesis (as measured by [(3)H]thymidine incorporation) in Ad GnRH-R-infected cells (but not in control cells). This effect was mimicked by agonist analogues and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at a density comparable to that in gonadotrophs, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotrophs. Moreover, expression of high affinity GnRH-Rs can facilitate a direct anti-proliferative effect of GnRH agonists on prostate cancer cells.

Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors.

Sustained stimulation of G-protein-coupled receptors (GPCRs) typically causes receptor desensitisation, which is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G protein (causing desensitisation), but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C (PLC)-coupled GPCRs on pituitary gonadotrophs to stimulate a Ca(2+)-mediated increase in gonadotrophin secretion. The type I GnRH receptors (GnRH-Rs), found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin; they are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation, with concomitant receptor phosphorylation (within the C-terminal tails) or binding of beta-arrestin, or both. The association with beta-arrestin may also be important for regulation of dynamin, a GTPase that controls separation of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-Rs in Hela cells conditionally expressing a dominant negative mutant of dynamin (K44A), we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (xenopus) GnRH-Rs but not type I (human) GnRH-Rs. In these cells, blockade of dynamin-dependent internalisation also inhibited GnRH-R-mediated MAPK activation, but this effect was not receptor specific and therefore not dependent upon dynamin-regulated GnRH-R internalisation. Although type I GnRH-Rs do not desensitise, sustained activation of GnRH-Rs causes desensitisation of gonadotrophin secretion, and we have found that GnRH can cause down-regulation of inositol (1,4,5) trisphosphate receptors and desensitisation of Ca(2+) mobilisation in pituitary cells. The atypical resistance of the GnRH-R to desensitisation may underlie its atypical efficiency at provoking this downstream adaptive response. GnRH-Rs are also expressed in several extrapituitary sites, and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R-expressing adenovirus facilitated expression of high-affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells, and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the mammalian GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-Rs in pituitary and extrapituitary sites.

The gonadotrophin-releasing hormone receptor: signalling, cycling and desensitisation.

Sustained stimulation of G-protein coupled receptors (GPCRs) typically causes receptor desensitisation that is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G-protein (causing desensitisation) but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C coupled GPCRs on pituitary gonadotrophs. The type I GnRH-receptors (GnRH-Rs) found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin. They are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation with concomitant receptor phosphorylation (within the C-terminal tails) and/or binding of beta-arrestin. The binding to beta-arrestin may also be important for association with dynamin, a GTPase that controls cleavage of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-R, we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (Xenopus) GnRH-Rs but not type I (human) GnRH-Rs, revealing the existence of functionally distinct routes through which these receptors are internalised. Although type I GnRH-R do not rapidly desensitise, sustained activation of GnRH receptors does cause desensitisation of gonadotrophin secretion, an effect which must therefore involve adaptive responses distal to the receptor. One such response is the GnRH-induced down regulation of inositol 1, 4, 5 trisphosphate receptors that apparently underlies desensitisation of Ca2+ mobilisation in a gonadotroph-derived cell line. Although activation of other GPCRs can down-regulate inositol 1, 4, 5 trisphosphate receptors, the effect of GnRH is atypically rapid and pronounced, presumably because of the receptor's atypical resistance to desensitisation. GnRH-Rs are also expressed in several extra-pituitary sites and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R expressing adenovirus facilitated expression of high affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-R signalling in pituitary and extra-pituitary sites.

Signaling and antiproliferative effects mediated by GnRH receptors after expression in breast cancer cells using recombinant adenovirus.

GnRH receptors (GnRH-Rs) are found in human cancers, including those of the breast, and GnRH can inhibit the growth of cell lines derived from such cancers. Although pituitary and extrapituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extrapituitary GnRH-Rs have low affinity for GnRH analogs and may not activate PLC or discriminate between agonists and antagonists in the same way as pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in breast cancer cells differ from those in gonadotropes. We found no evidence for endogenous GnRH-Rs in MCF7 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at a multiplicity of infection of 10 or greater, at least 80% expressed GnRH-Rs. These had high affinity (K(d) for [(125)I]buserelin, 1.4 nM) and specificity (rank order of potency, buserelin>GnRH>>chicken GnRH-II) and mediated stimulation of [(3)H]IP accumulation. Increasing viral titer [from multiplicity of infection, 3-300] increased receptor number (10,000-225,000 sites/cell) and [(3)H]IP responses. GnRH stimulated ERK2 phosphorylation in Ad GnRH-R-infected cells, and this effect, like stimulation of [(3)H]IP accumulation, was blocked by GnRH-R antagonists. GnRH also inhibited [(3)H]thymidine incorporation into Ad GnRH-R-infected cells (but not control cells). This effect was mimicked by agonist analogs and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at density comparable to that in gonadotropes, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotropes, and increasing expression of high affinity GnRH-Rs can dramatically enhance the direct antiproliferative effect of GnRH agonists on breast cancer cells.

Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling.

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.

Aplysia ror forms clusters on the surface of identified neuroendocrine cells.

The ror receptors are a highly conserved family of receptor tyrosine kinases genetically implicated in the establishment of cellular polarity. We have cloned a ror receptor from the marine mollusk Aplysia californica. Aplysia ror (Apror) is expressed in most developing neurons and some adult neuronal populations, including the neuroendocrine bag-cell neurons. The Apror protein is present in peripheral neuronal processes and ganglionic neuropil, implicating the kinase in the regulation of growth and/or synaptic events. In cultured bag-cell neurons, the majority of the protein is stored in intracellular organelles, whereas only a small fraction is expressed on the surface. When expressed on the cell surface, the protein is clustered on neurites, suggesting that Apror is involved in the organization of functional domains within neurons. Apror immunoreactivity partially colocalizes with the P-type calcium channel BC-alpha1A at bag-cell neuron varicosities, suggesting a role for Apror in organizing neuropeptide release sites.

Desensitization and internalization of human and xenopus gonadotropin-releasing hormone receptors expressed in alphaT4 pituitary cells using recombinant adenovirus.

Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using alphaT4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in alphaT4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH3 cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.

The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alphaT3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size.

The phospholipase C (PLC)-activating gonadotrophin-releasing hormone (GnRH) receptor is thought not to rapidly desensitise in alphaT3-1 cells. This extremely unusual characteristic raises the concern that it might be a feature of the cell type, rather than the receptor per se. Here we have used video imaging to establish whether the effects of endogenous PLC-activating G-protein coupled receptors (GPCRs) on Ca2+ ion concentration [Ca2+]i desensitise in these cells. Oxytocin, endothelin-1, methacholine, and UTP all caused [Ca2+]i increases which underwent rapid homologous desensitisation in that they were transient and responses to repeat stimuli were attenuated whereas subsequent responses to GnRH were not. To test whether receptor reserve obscures functional desensitisation of GnRH receptors, a photoaffinity antagonist (Pant-1), was used to effect a partial and irreversible receptor blockade. UV crosslinking in medium with 1000 nM Pant-1 reduced GnRH receptor number to 20 +/- 5% and reduced maximal buserelin-stimulated [3H]IP(X) accumulation to 57 +/- 5%, demonstrating removal of receptor reserve. In control alphaT3-1 cells the initial rate of GnRH-stimulated [3H]IP(X) accumulation was maintained for at least 5 min and GnRH caused a sustained increase in Ins(1,4,5)P3 mass (confirming the resistance of GnRH receptors to desensitisation) and Pant-1 pre-treatment reduced the magnitude of these responses without altering their temporal profiles. In alphaT3-1 cells stably transfected with recombinant human muscarinic receptors (alphaT3-1/M3), responses to methacholine were characteristic of desensitising GPCRs (transient Ins(1,4,5)P3 and curvilinear [3H]IP(X) responses) and were unaltered by Pant-1. To test the relevance of phospholipid pool size, alphaT3-1/M3 cells were pre-treated with GnRH or methacholine in medium with LiCl (to deplete PtdIns(4,5)P2 pools). These pre-treatments reduced subsequent responses to methacholine and GnRH comparably, indicating access to a shared PtdIns(4,5)P2 pool. Partial depletion of this pool (GnRH pre-treatment in medium with LiCl) reduced the magnitude of the [3H]IP(X) and Ins(1,4,5)P3 responses to methacholine and GnRH, without altering their temporal profiles. Thus the GnRH receptor does not undergo rapid homologous desensitisation in alphaT3-1 cells in spite of the fact that they can desensitise other endogenous (and recombinant) PLC-activating GPCRs, and the lack of desensitisation cannot be attributed to the existence of GnRH receptor reserve or access to an atypically large or rapidly re-cycled PtdIns(4,5)P2 pool. This unique functional characteristic (mammalian GnRH receptors are the only PLC-activating GPCRs known not to rapidly desensitise) almost certainly therefore reflects the atypical structure of these receptors (mammalian GnRH receptors are the only PLC-activating GPCRs known to lack C-terminal tails).

The implementation of an integrated on-line health education system at RMIT.

The Faculty of Biomedical and Health Sciences at RMIT has been developing an on-line health education system using a systems thinking approach, to create a learning environment whose basis is supported by Information Technology (IT). The centre-piece of this system is the Faculty Learning Centre, which has been created, both in space and layout, to promote collaborative learning between the students, so that the educator is physically assimilated with the student body. This facility is supplemented by the Faculty WWW server, which has been the main vehicle for course material dissemination to students. To ensure an effective on-line teaching environment, the position of an on-line facilitator has been created, whose responsibilities include both the continual evaluation of the system and the implementation of appropriate system changes. Aspects have included the production of a staff development training program and extensive user documentation. This paper discusses the systems thinking approach used to implement this integrated on-line system, and the establishment of explicit educational rationales in the use of IT to support learning strategies. Some examples of the on-line educational programs are also presented.

Biochemical pathways by which serotonin regulates translation in the nervous system of Aplysia.

In the marine mollusk Aplysia californica, serotonin initiates three phases of translational regulation: an initial decrease in translation, followed by a transient increase in protein synthesis, both of which are independent of transcription, followed by a later increase in protein synthesis that is dependent on transcription. These increases in protein synthesis may underlie translation-dependent changes in synaptic plasticity. We have characterized the second messenger pathways that underlie these changes in the pleural ganglia of Aplysia. Activation of protein kinase C was both necessary and sufficient for the initial decrease in translation. Protein kinase C, cyclic AMP-dependent protein kinase, and a tyrosine kinase were all required for the second phase, a transient increase in protein synthesis. The late increase in protein synthesis required both protein kinase A and spaced applications of serotonin. Rapamycin, a specific inhibitor of a downstream translational regulator, blocked the transient increase in protein synthesis (second phase), suggesting that this drug may be useful in determining the specific physiological consequences of this translational regulation. Indeed, we used rapamycin to demonstrate that one type of intermediate form of synaptic plasticity induced by serotonin did not require the rapamycin-sensitive increase in translation.

Elevation of lead in human blood from its controlled ingestion in beer.

1. Nine volunteers ingested high levels of lead in beer over a 28-d period. The increase in blood lead varied by a factor of about two. There was a similar two-fold variability in the whole-body uptake (mean 14%) of a single oral dose of the short-lived tracer 203Pb. 2. The average elevations led to estimates of the potential increment from consumption of alcoholic beverages which accord broadly with epidemiological observation and which, if relevant to intakes of lead in table wine, raise the possibility of considerably elevated levels in the blood of avid consumers. 3. Rate constants inferred for removal of stable lead from blood were lower than reported following intake of the tracer, reflecting feedback of lead from other compartments.

Genetic homogeneity between acute and chronic forms of spinal muscular atrophy.

The childhood-onset spinal muscular atrophies (SMAs) describe a heterogeneous group of disorders that selectively affect the alpha motoneuron. We have shown that chronic childhood-onset SMA (SMA II and III) maps to a single locus on chromosome 5q. Acute SMA (SMA Type I/Werdnig-Hoffmann/severe/infantile) is the main cause of heritable infant mortality. Mapping the acute SMA locus by conventional methods is complicated by the rapidly fatal course of the disease and its recessive mode of inheritance. We present here the typing of four inbred acute-SMA families with DNA markers on chromosome 5q and analysis of these together with acute families from our previous study to demonstrate genetic homogeneity between the acute and chronic forms of SMA. The data indicate that the acute SMA locus maps to chromosome 5q11.2-13.3. Two families seem unlinked to 5q markers, raising the possibility of genetic heterogeneity or disease misclassification within the acute and chronic family sets.

Outcome of infants shunted for post-haemorrhagic ventricular dilatation.

Between April 1980 and March 1986, 19 infants underwent cerebrospinal fluid (CSF) shunting procedures for post-haemorrhagic ventricular dilatation at the Hammersmith Hospital, London. A total of 58 shunt-related procedures have been performed on these children. The major perioperative complication was seizure activity (eight children). Postoperative complications included infection (12 shunts) and blockage (29 shunts). Prophylactic antibiotics failed to prevent shunt infection. The likelihood of the first shunt failing was significantly reduced by greater weight of the infant and lower CSF protein at surgery. Long-term outcome was poor: three have died and another four are quadriplegic with severe mental retardation. Only four children are developmentally normal. These outcomes cannot be related to the shunt surgery or its complications, but correlate best with pre-operative parenchymal brain-lesions, as shown on ultrasound scans.

Amino acid profiles during development of the fetal rat.

Umbilical blood venous-arterial differences for amino acids across the rat fetus show uptake of disproportionately large amounts of glutamine, alanine and lysine relative to their contribution to total body protein. Free amino acid concentrations in fetal liver tend to increase during development, but show a rapid adjustment to near-adult values within 24 h after birth.