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This new English edition of Arnold Geulincx’ Ethics is the first complete edition to appear in a modern language and includes notes by the great Irish writer Samuel Beckett, who indicated that Geulincx was a key influence on his works.

This new English edition of Arnold Geulincx’ Ethics is the first complete edition to appear in a modern language and includes notes by the great Irish writer Samuel Beckett, who indicated that Geulincx was a key influence on his works.

The Ca2+ that promotes transmitter release is generally thought to enter presynaptic terminals through voltage-gated Ca2+channels. Using electrophysiology and Ca2+ imaging, we show that, in amacrine cell dendrites, at least some of the Ca2+ that triggers transmitter release comes from endoplasmic reticulum Ca2+ stores. We show that both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) are present in these dendrites and both participate in the elevation of cytoplasmic [Ca2+] during the brief depolarization of a dendrite. Only the Ca2+ released through IP3Rs, however, seems to promote the release of transmitter. Antagonists for the IP3R reduced transmitter release, whereas RyR blockers had no effect. Application of an agonist for metabotropic glutamate receptor, known to liberate Ca2+ from internal stores, enhanced both spontaneous and evoked transmitter release.

It has been suggested that multiple sclerosis (MS) patients with positive anticardiolipin antibodies (ACLA) have some atypical features, including absent oligoclonal bands (OCB) in the cerebrospinal fluid (CSF). Our aim was to compare the frequencies of ACLA and related laboratory and clinical features in OCB negative (OCB−) and positive (OCB+) MS patients. We compared 41 OCB− patients attending a MS Clinic in a tertiary referral center, with 206 OCB+ patients. ACLA, anti-b2-glycoprotein and other autoantibodies, lupus anticoagulant and coagulation markers were measured. We found a higher frequency of ACLA in OCB− patients, 18/41 versus 33/206 in OCB+ patients (P < 0.0001). OCB− patients had more progressive MS than OCB+ subjects. There were no differences in age, sex, Expanded Disability Status Scale (EDSS) score, antiphospholipid syndrome symptoms between the groups. ACLA+ MS patients were more frequently in the OCB− group. Although this may suggest that they represent a special subgroup of MS, no other clinical or laboratory findings distinguish the groups. Although OCB− MS patients may be thought to be less active immunologically, this study shows they have more frequently ACLA than OCB+ patients. OCB− MS patients in our cohort do not appear to have a more benign form of MS, as has previously been suggested.

We report the case of a 66-year-old black woman who presented with concomitant acute infectious keratitis, bacteremia, and septic arthritis caused by Streptococcus pneumonia. The septic arthritis resolved rapidly with surgical drainage and intravenous antibiotics, but despite aggressive topical and intravenous antibiotic therapy for the infectious keratitis, the cornea perforated, the patient developed endophthalmitis, and the eye eventually was eviscerated. To the best of our knowledge this is the first reported case of this nature. This patient had undergone splenectomy > 50 years prior to developing these infections. Although the risk of serious infection in clinically significant bacteremia is greatest in the perioperative period after splenectomy, these patients are at increased risk of such events for a lifetime. Because encapsulated bacteria, especially Pneumococcus, pose the greatest risk of sepsis and infection in asplenic patients, pneumococcal vaccination of penicillin prophylaxis must always be considered in these patients. A careful and complete medical history and systemic evaluation remain a crucial element of the evaluation and management of serious infectious keratitis.

The adverse health effects of elevated exposures to PM(10) (particulate matter collected through a size selective inlet with an efficiency of 50% for particles with an aerodynamic diameter of 10 μm) in relation to morbidity and mortality, especially in susceptible individuals, are now well recognised. PM(10) consists of a variable cocktail of components differing in chemical composition and size. Epidemiological and toxicological data suggest that transition metals and ultrafine particles are both able to drive the cellular and molecular changes that underlie PM(10)-induced inflammation and so worsen disease status. Toxicological evidence also suggest roles for the biological components of PM(10) including volatile organic compounds (VOC's), allergens and bacterial-derived endotoxin. Many of these components, in particular transition metals, ultrafine particles, endotoxin and VOC's induce a cellular oxidative stress which initiates an intracellular signaling cascade involving the activation of phosphatase and kinase enzymes as well as transcription factors such as nuclear factor kappa B. Activation of these signaling mechanisms results in an increase in the expression of proinflammatory mediators, and hence enhanced inflammation. Given that many of the components of PM(10) stimulate similar or even identical intracellular signaling pathways, it is conceivable that this will result in synergistic or additive interactions so that the biological response induced by PM(10) exposure is a response to the composition rather than the mass alone. A small number of studies suggest that synergistic interactions occur between ultrafine particles and transition metals, between particles and allergens, and between particles and VOC's. Elucidation of the consequences of interaction between the components of PM(10) in relation to their biological activity implies huge consequences for the methods used to monitor and to legislate pollution exposure in the future, and may drive a move from mass based measurements to composition.

Both the ultrafine particle and transition metal components of particulate air pollution (PM(10)) have been hypothesized to be important factors in determining toxicity and potential adverse health effects. In this study we aimed to investigate interactions between transition metal salts and a surrogate environmental particle-ultrafine carbon black (ufCB). In all experimental systems employed, the ufCB was found to be more reactive than its fine counterpart (CB). Incubation of ufCB with the reactive oxygen species (ROS)-sensitive probe dichlorofluorescin in the absence of cells generated significantly more ROS than CB. With addition of either cupric sulfate (CuSO(4)), ferrous sulfate (FeSO(4)), or ferric chloride (FeCl(3)), the ROS generation in the presence of ufCB was enhanced in a potentiative manner. In Mono Mac 6 macrophages, ufCB again produced more ROS than CB. However, addition of iron salts had no additive effect over and above that induced in the macrophages by ufCB. In the mouse macrophage cell line J774, ufCB decreased the cellular content of GSH and ATP. Addition of iron further decreased both GSH and ATP and a potentiative interaction between ufCB and FeSO(4) was observed, but only at the highest iron concentrations tested. A concentration-dependent increase in tumor necrosis factor-alpha production by J774 cells was also observed following exposure to ufCB, which was not further enhanced by the addition of iron. J774 cells were also found to sequester or chelate iron without inducing toxicity. In the rat lung ufCB induced a significant neutrophil influx and this inflammatory effect was potentiativelly enhanced by the addition of FeCl(3) (100 microM). These findings suggest that (1) ultrafine particles and metals interact by chemical potentiation in a cell-free environment to generate ROS, (2) potentiation between ultrafine particles and metal salts is not observed in the presence of macrophages as iron is sequestered or chelated by the cells, (3) in the lung, ultrafine particles and iron salts interact in a potentiative manner to generate inflammation.

We have previously shown that disabling forward-mode Na+-Ca2+exchange in amacrine cells greatly prolongs the depolarization-induced release of transmitter. To investigate the mechanism for this, we imaged [Ca2+]iin segments of dendrites during depolarization. Removal of [Na+]oproduced no immediate effect on resting [Ca2+]ibut did prolong [Ca2+]itransients induced by brief depolarization in both voltage-clamped and unclamped cells. In some cells, depolarization gave rise to stable patterns of higher and lower [Ca2+] over micrometer-length scales that collapsed once [Na+]owas restored. Prolongation of [Ca2+]itransients by removal of [Na+]ois not due to reverse mode operation of Na+-Ca2+exchange but is instead a consequence of Ca2+release from endoplasmic reticulum (ER) stores over which Na+-Ca2+exchange normally exercises control. Even in normal [Na+]o, hotspots for [Ca2+] could be seen following depolarization, that are attributable to local Ca2+-induced Ca2+release. Hotspots were seen to be labile, probably reflecting the state of local stores or their Ca2+release channels. When ER stores were emptied of Ca2+by thapsigargin, [Ca2+] transients in dendrites were greatly reduced and unaffected by the removal of [Na+]oimplying that even when Na+-Ca2+exchange is working normally, the majority of the [Ca2+]iincrease by depolarization is due to internal release rather than influx across the plasma membrane. Na+-Ca2+exchange has an important role in controlling [Ca2+] dynamics in amacrine cell dendrites chiefly by moderating the positive feedback of the Ca2+amplifier.

What do most people think of a law degree – plentiful amounts of hard work, endless reading, an expensive Legal Practice Course or Bar Vocational Course followed by an extremely competitive application process to secure a job in the graduate’s respective field of work? What if students were given the opportunity to work in another jurisdiction, such as Australia, for one month in a student-run law office during the summer, with £1000 to get them on their way? ‘Sounds good’, I thought. ‘So what do I have to do to get the chance’? And then the one catch is divulged – the student must complete a compulsory piece of coursework and achieve one of the top 10 marks in their year. (The writer expects a few raised eyebrows at this stage!)

What do most people think of a law degree – plentiful amounts of hard work, boundless reading, an expensive Legal Practice Course or Bar Vocational Course followed by an extremely competitive application process to secure a job in the graduate’s respective field of work? What if students were given the opportunity to work in another jurisdiction, such as Australia, for one month in a student run law office, during the summer, with £1000 to get them on their way? ‘Sounds good’, I thought. ‘So what do I have to do to get the chance’? And then the one catch is divulged – the students must complete a compulsory piece of coursework and achieve one of the top 10 marks in their year. (The writer expects a few raised eyebrows at this stage!)

Studies into the effects of ultrafine particles in the lung have shown adverse effects considered to be due in part to the particle size. Air pollution particles (PM(10)) are associated with exacerbations of respiratory disease and deaths from cardiovascular causes in epidemiological studies and the ultrafine fraction of PM(10) has been hypothesized to play an important role. The aim of the present study was to investigate proinflammatory responses to various sizes of polystyrene particles as a simple model of particles of varying size including ultrafine. In the animal model, we demonstrated that there was a significantly greater neutrophil influx into the rat lung after instillation of 64-nm polystyrene particles compared with 202- and 535-nm particles and this was mirrored in other parameters of lung inflammation, such as increased protein and lactate dehydrogenase in bronchoalveolar lavage. When surface area instilled was plotted against inflammation, these two variables were directly proportional and the line passed through zero. This suggests that surface area drives inflammation in the short term and that ultrafine particles cause a greater inflammatory response because of the greater surface area they possess. In vitro, we measured the changes in intracellular calcium concentration in mono mac 6 cells in view of the potential role of calcium as a signaling molecule. Calcium changes after particle exposure may be important in leading to proinflammatory gene expression such as chemokines. We demonstrated that only ultrafine polystyrene particles induced a significant increase in cytosolic calcium ion concentration. Experiments using dichlorofluorescin diacetate demonstrated greater oxidant activity of the ultrafine particles, which may explain their activity in these assays. There were significant increases in IL-8 gene expression in A549 epithelial cells after treatment with the ultrafine particles but not particles of other sizes. These findings suggest that ultrafine particles composed of low-toxicity material such as polystyrene have proinflammatory activity as a consequence of their large surface area. This supports a role for such particles in the adverse health effects of PM(10).

OBJECTIVES—In July 1995 the Soufriere Hills volcano on the island of Montserrat began to erupt. Preliminary reports showed that the ash contained a substantial respirable component and a large percentage of the toxic silica polymorph, cristobalite. In this study the cytotoxicity of three respirable Montserrat volcanic ash (MVA) samples was investigated: M1 from a single explosive event, M2 accumulated ash predominantly derived from pyroclastic flows, and M3 from a single pyroclastic flow. These were compared with the relatively inert dust TiO2 and the known toxic quartz dust, DQ12. METHODS—Surface area of the particles was measured with the Brunauer, Emmet, and Teller (BET) adsorption method and cristobalite content of MVA was determined by x ray diffraction (XRD). After exposure to particles, the metabolic competence of the epithelial cell line A549 was assessed to determine cytotoxic effects. The ability of the particles to induce sheep blood erythrocyte haemolysis was used to assess surface reactivity. RESULTS—Treatment with either MVA, quartz, or titanium dioxide decreased A549 epithelial cell metabolic competence as measured by ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). On addition of mannitol, the cytotoxic effect was significantly less with M1, quartz, and TiO2. All MVA samples induced a dose dependent increase in haemolysis, which, although less than the haemolysis induced by quartz, was significantly greater than that induced by TiO2. Addition of mannitol and superoxide dismutase (SOD) significantly reduced the haemolytic activity only of M1, but not M2 or M3, the samples derived from predominantly pyroclastic flow events. CONCLUSIONS—Neither the cristobalite content nor the surface area of the MVA samples correlated with observed in vitro reactivity. A role for reactive oxygen species could only be shown in the cytotoxicity of M1, which was the only sample derived from a purely explosive event. These results suggest that in general the bioreactivity of MVA samples in vitro is low compared with pure quartz, but that the bioreactivity and mechanisms of biological interaction may vary according to the ash source. Keywords: volcanic ash; cristobalite; surface reactivity

The concept of a safe, immunocontraceptive vaccine using the zona pellucida glycoprotein 3 (ZP3) as an immunogen has been marred by the appearance of ovarian dysfunction in several species. However, careful selection of epitopes on mouse ZP3 have demonstrated that it is possible to segregate contraceptive bone marrow-derived (B)-cell epitopes from the cytotoxic thymus-derived (T)-cell epitopes thought to be responsible for inducing ovarian disease. B-cell epitopes on marmoset ZP3 (mstZP3) were identified by epitope mapping studies. Using a panel of polyclonal antibodies against recombinant mstZP3, an immunodominant epitope mstZP3(301-320) was identified. A chimeric peptide was co-linearly synthesized incorporating this sequence with a promiscuous tetanus toxoid T-helper cell epitope. Using the common marmoset (Callithrix jacchus) as an animal model, we have compared the consequences of active immunization with homologous recombinant mstZP3 and mstZP3(301-320) chimeric peptide vaccine. Long-term infertility was achieved using mstZP3 but at the expense of ovarian function. In contrast, no disruption to ovarian function was observed following mstZP3(301-320) immunization. Antibodies to this peptide immunolocalized to the zona pellucida of both marmoset and human ovarian sections and inhibited human sperm-zona binding by approximately 60% in vitro. However, in-vivo studies indicated that targeting a single ZP3 epitope was insufficient to reliably and consistently achieve a contraceptive effect.

PROBLEM: The unique recognition events that result in the avid binding of mammalian spermatozoa to the surface of the zona pellucida (ZP) are being exploited in the development of contraceptive vaccines. In this study, the safety and efficacy of a vaccination strategy based on the induction of active immunity against purified, glycosylated, recombinant human ZP3 (rhZP3) has been evaluated in a primate model, Callithrix jacchus. METHOD OF STUDY: Long-term infertility was established after immunization with rhZP3 and the resulting immune sera reacted with rhZP3 on an enzyme-linked immunosorbent assay (ELISA) and immunolocalized exclusively to the outer surface of native ZP on marmoset ovarian sections. However, this contraceptive effect was inevitably associated with the eventual appearance of an ovarian pathology characterized by a depletion of primordial follicles. In an attempt to circumvent this side effect, human ZP3 (hZP3) was epitope mapped and four continuous, immunodominant B-cell epitopes (hZP3 45-64 , hZP3 93-110 , hZP3 172-190 and hZP3 341-360 ) were evaluated for contraceptive efficacy in vivo. Using peptide-tetanus toxoid (TT) conjugates to enhance immunogenicity, antipeptide antibodies were raised against these immunogens, which also cross-reacted with rhZP3 on ELISA. In addition, antibodies against hZP3 45-64 and hZP3 172-190 recognized native ZP on marmoset ovarian sections when a microwave technique was used to enhance epitope presentation. RESULTS: No ovarian pathology was observed after the long-term administration of these peptide immunogens, and fertility was suppressed when compared with TT controls but could not be correlated to the antibody titer. CONCLUSION: Clearly, further research is required to identify optimal B-cell epitopes that will reliably induce infertility, free from any ovarian pathology.

Frerking, Matthew, Salvador Borges, and Martin Wilson. Are some minis multiquantal? J. Neurophysiol. 78: 1293–1304, 1997. The amplitude distribution of miniature postsynaptic currents (minis) in many central neurons has a large variance and positive skew, but the sources of this variance and skew are unresolved. Recently it has been proposed that spontaneous Ca2+ influx into a presynaptic bouton with multiple release sites could cause spontaneous multiquantal minis by synchronizing release at all sites in the bouton, accounting for both the large variance and skew of the mini distribution. We tested this hypothesis by evoking minis with internally perfused, buffered Ca2+ and the secretagogue α-latrotoxin, both in the absence of external Ca2+. With these manipulations, the synchronized release model predicts that the mini distribution should collapse to a Gaussian distribution with a reduced coefficient of variation. Contrary to this expectation, we find that mini amplitude distributions under these conditions retain a large variance and positive skew and are indistinguishable from amplitude distributions of depolarization-evoked minis, strongly suggesting that minis are uniquantal.

Introduction Methyl Iodide is well recognised as an industrial toxin and accidental exposure at workplace can cause neuropsychiatric symptoms. 1 We report two cases with similar clinical presentations, and previously unreported MRI abnormalities. Case 1 A 44 year old gentleman developed visual hallucinations on his first day back from work at a pharmaceutical factory. Later he became extremely agitated and psychotic with paranoid delusions and auditory hallucinations and also developed difficulty walking. A history of possible inhalation of a gas was obtained from a colleague at workplace. Examination on admission showed nystagmus, cerebellar ataxia, asterixis and marked confusion. An MRI scan done few days after admission showed subtle signal change in the posterior fossa and CSF examination was normal. Routine bloods were normal, but Blood and urine Iodine levels were more than 100 times above the normal range. His symptoms gradually settled on observation and a small dose of antipsychotic over three weeks, though he has been left with mild behavioural changes. Case 2 A 40 year old man reported accidentally inhaling Methyl Iodide at workplace and developed walking difficulties, visual hallucinations and repeated seizures over the next 12 hours. He was admitted in status epilepticus to the local hospital and was had to be sedated and intubated as his seizures remained uncontrolled with intravenous bolus doses of Phenytoin and Sodium Valproate. Examination after weaning off sedation showed marked dysarthria, cerebellar ataxia and he also reported delusional ideas and olfactory hallucinations. MRI scans showed diffuse white matter abnormalities in the posterior fossa involving brainstem and cerebellum (similar to case 1) and routine blood and CSF investigations were normal, but blood and urine Iodine levels were more than 500 times the normal range. His symptoms gradually improved and was discharged home 8 weeks after his initial presentation, but on follow up two months later, he still needed crutches to mobilise and had persistent olfactory hallucinations. Discussion Though Neuropsychiatric features are well recognised in Methyl Iodide toxicity in the past, abnormalities on brain imaging have not been previously reported. Both of our cases presented a distinct clinical and radiological picture with encephalopathy, ataxia and neuropsychiatric symptoms, with similar changes in the posterior fossa on MRI. Case 2 had a more severe clinical presentation and more significant scan changes and this is possibly because he had a more severe exposure as evidenced by the higher levels of Iodine in blood and urine. Conclusion Methyl Iodide poisoning should be considered in the appropriate clinical setting when patients present with encephalopathy, ataxia and abnormalities in the posterior fossa which can sometimes be mistaken for acute demyelination. Obtaining history of exposure is the key to the diagnosis and avoiding unecessary investigations, emphasising the importance of occupational history in these cases.

A fundamental issue in synaptic physiology is whether the postsynaptic response to a quantum of transmitter is limited by the number of receptors available. Fierce debate over the past few years has yielded no consensus. The majority of evidence suggests that the degree of receptor occupancy is likely to be sensitive to a number of factors, including the detailed anatomy of the synaptic cleft and the time course of transmitter clearance, and is probably different from one synapse to the next.

The strength of synaptic connections between two neurons is characterized by the number of release sites (N) on the presynaptic cell, the probability (p) of transmitter release at those sites in response to a stimulus, and the average size (A) of the postsynaptic response from each site. Quantal analysis can determine N, p, and A, but the large variance in the amplitudes of minis at central synapses is predicted to obscure quantal peaks and render quantal analysis unusable. Recently it has been suggested that the variance in mini amplitude is generated by differences between release sites, rather than by quantum-to-quantum fluctuations at identical sites, and that this form of variance in mini amplitude reduces the amount of variance expected in quantal peaks. Using simulations, we examine the possibility of resolving quantal peaks assuming either form of variance in mini amplitude. We find that individual quantal peaks are resolvable in neither case, provided that the uniquantal distribution is similar to the mini distribution. Because this lack of resolution compromises the utility of quantal analysis, we develop a general description that can solve N and p, given the statistical parameters of the mini distribution and the evoked distribution. We find that this description is relatively insensitive to the source of variance in mini amplitude.

The peptide, neurotensin, is found in a class of amacrine cells synapsing chiefly withother amacrine cells in the chicken retina (Li & Lam, 1990; Watt et al., 1991). Toinvestigate the possible effects of neurotensin, we have used Ca2+imaging to measure cytosolic Ca2+concentrations in cultured chick amacrine cells. Following a delay of about 2 min, neurotensin (300 nM) induced oscillations in Ca2+concentration that typically had a period of 2 min and peak values of about 300 nM when averaged over the cell body. The phospholipase C inhibitors U-73, 112 and 4′-bromophenacyl bromide terminated oscillations induced by neurotensin but the protein kinase inhibitors H7 and staurosporine did not inhibit oscillations, increasing their frequency instead. In the absence of external Ca2+, neurotensin induced only a single Ca2+transient, much briefer than when external Ca2+was present. Together these results suggest that neurotensin activates phospholipase C, thereby producing IP3that triggers Ca2+release from an internal store. Although this released Ca2+contributes to periodic Ca2+peaks, the majority of cytosolic Ca2+, even in the first peak, comes from Ca2+influx across the plasmalemma.

Do graded potential synapses work the same way as action potential synapses? Recent work emphasizes the differences and suggests that graded potential synapses are not all the same either.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Calcium influx into cultured retinal amacrine cells is followed by a small, slow, inward current that we show here results from the operation of electrogenic Na-Ca exchange. The activity of the exchanger is shown to correlate with the magnitude of the Ca2+ load and to depend on both the Ca2+ and Na+ gradients. Li+ is unable to substitute for Na+ and in the absence of Na+, slow tail currents are almost entirely suppressed. A rapid change in [K+]o does not affect the activity of the exchanger, suggesting that only Na+ and Ca2+ are transported. The ratio of charge entering as Ca2+ current to the charge entering as exchange current is highly variable between cells. We suggest that variability results from a variable fraction of Ca2+ load, we estimate typically 40%, being removed by a process other than Na-Ca exchange. This process is likely to involve internal buffering or sequestration since inhibition of the plasmalemmal Ca(2+)-ATPase does not increase the fraction of Ca2+ expelled by the exchanger. Ca2+ loading performed in the absence of Na+o generates smaller exchange charge the longer the delay in returning Na+o to the neuron. About 30% of exchange charge is lost for a delay of 1 sec.

(1)H nuclear magnetic resonance spectroscopy (NMR) resonances from lipids in tumours are associated with tumour grade and treatment response. The origin of these NMR signals is mainly considered to be cytoplasmic lipid droplets (LDs). Techniques exist for isolating LDs but little is known about their composition and its relationship to NMR signals. In this work, density-gradient ultracentrifugation was performed on homogenised human cancer cells to isolate LDs. (1)H NMR was performed on whole cells, isolated LDs and their extracts. Heteronuclear single quantum coherence spectroscopy (HSQC) and liquid chromatography mass spectroscopy (LC-MS) were performed on lipid extracts of LDs. Staining and microscopy were used to characterize isolated LDs. An excellent agreement in chemical shift and relative signal intensity was observed between lipid resonances in cells and isolated LD spectra supporting that NMR-visible lipids originate primarily from LDs. Isolated LDs showed high concentrations of unsaturated lipids, a oleic-to-linoleic acid ratio greater than two and a cholesteryl ester (ChE)-to-cholesterol (Ch) ratio close to unity. These ratios were several-fold greater than respective ratios in whole cells, demonstrating isolation is important to characterize LD composition. LDs contain a specific group of lipid species that are likely to contribute to the (1)H NMR spectrum of cells.

Cultured retinal amacrine cells show quantal GABAergic synaptic transmission. Voltage clamping pre- and post-synaptic cells of an isolated pair has allowed us to examine the entry and removal of Ca2+ at synaptic terminals. Brief presynaptic Ca2+ currents elicit an initial postsynaptic current that probably reflects the roughly synchronous exocytosis of docked vesicles. Prolonged Ca2+ currents elicit an additional second phase of release whose time course can greatly exceed that of the presynaptic voltage step. The time course of this second phase reflects a sustained increase in cytosolic Ca2+ and is matched closely by the activity of the presynaptic Na-Ca exchanger, as revealed by an exchange current. Eliminating the activity of the exchanger by removal of external Na+ prolongs this second phase of transmission greatly. Because transmitter release at these synapses outlasts Ca+ channel opening, Na-Ca exchange plays a significant role in shaping transmission.

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 x B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by the bar or pat genes, was more efficient than selection on kanamycin, mediated by the nptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants--transgene silencing, as well as poor transgene transmission to progeny was observed in some plant lines in which the parent plants had expressed the transgene.

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.