Intracellular trafficking mechanism of cationic phospholipids including cationic liposomes in HeLa cells.

The development of gene delivery methods is essential for the achievement of effective gene therapy. Elucidation of the intracellular transfer mechanism for cationic carriers is in progress, but there are few reports regarding the intracellular trafficking processes of the cationic phospholipids taken up into cells. In the present work, the trafficking processes of a cationic phospholipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) were investigated from intracellular uptake to extracellular efflux using cationic liposomes in vitro. Following intracellular transport of liposomes via endocytosis, DOTAP was localized in the endoplasmic reticulum, Golgi apparatus, and mitochondria. Moreover, the proteins involved in DOTAP intracellular trafficking and extracellular efflux were identified. In addition, helper lipids of cationic liposomes were found to partially affect this intracellulartrafficking. These findings might provide valuable information for designing cationic carriers and avoiding unexpected toxic side effects derived from cationic liposomal components.

ß3 integrin is dispensable for conditioned fear and hebbian forms of plasticity in the hippocampus.

Integrins play key roles in the developing and mature nervous system, from promoting neuronal process outgrowth to facilitating synaptic plasticity. Recently, in hippocampal pyramidal neurons, ß3 integrin (ITGß3) was shown to stabilise synaptic AMPA receptors (AMPARs) and to be required for homeostatic scaling of AMPARs elicited by chronic activity suppression. To probe the physiological function for ITGß3-dependent processes in the brain, we examined whether the loss of ITGß3 affected fear-related behaviours in mice. ITGß3-knockout (KO) mice showed normal conditioned fear responses that were similar to those of control wild-type mice. However, anxiety-like behaviour appeared substantially compromised and could be reversed to control levels by lentivirus-mediated re-expression of ITGß3 bilaterally in the ventral hippocampus. In hippocampal slices, the loss of ITGß3 activity did not compromise hebbian forms of plasticity--neither acute pharmacological disruption of ITGß3 ligand interactions nor genetic deletion of ITGß3 altered long-term potentiation (LTP) or long-term depression (LTD). Moreover, we did not detect any changes in short-term synaptic plasticity upon loss of ITGß3 activity. In contrast, acutely disrupting ITGß1-ligand interactions or genetic deletion of ITGß1 selectively interfered with LTP stabilisation whereas LTD remained unaltered. These findings indicate a lack of requirement for ITGß3 in the two robust forms of hippocampal long-term synaptic plasticity, LTP and LTD, and suggest differential roles for ITGß1 and ITGß3 in supporting hippocampal circuit functions.

Zimmermann-Laband syndrome: a case report.

Zimmermann-Laband syndrome is a very rare disorder characterized by gingival fibromatosis, abnormalities of soft cartilages of the nose and/or ears, hypoplastic or absent nails and terminal phalanges, joint hypermobility, hypatoslenomegaly, mild hirsutism and learning difficulties. Early presentation of Zimmermann-Laband syndrome in a newborn has rarely been described. This paper describes a newborn patient with Zimmermann-Laband syndrome.

Calcium regulation of neurotransmitter release: reliably unreliable?

Recent studies of central synaptic transmission reveal that neurotransmitter release is more unreliable than was previously thought. Nerve stimulation does not always elicit transmitter release, and when release events occur vesicle fusion with the presynaptic membrane is limited to at most a single quantum.

Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro.

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.

Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro.

Mannose 6-phosphate receptors carry newly synthesized lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes and then return to the TGN to carry out another round of lysosomal enzyme delivery. Although clathrin-coated vesicles mediate the export of mannose 6-phosphate receptors from the TGN, nothing is known about the transport vesicles used to carry these receptors back to the TGN. Two different in vitro assays used in this study show that an antibody that interferes with clathrin assembly blocks receptor-mediated endocytosis of transferrin, but has no effect on the recycling of the 300-kilodalton mannose 6-phosphate receptor from prelysosomes to the TGN. These results suggest that the transport of mannose 6-phosphate receptors from prelysosomes to the TGN does not involve clathrin.

Long-term depression properties in a simple system.

Long-term depression (LTD) in pairs of cultured rodent hippocampal neurons was examined to study the molecular basis of this form of synaptic plasticity. We have previously characterized two components of transmitter release: a synchronous, fast phase that requires synaptotagmin I, and an asynchronous, slow component that persists in the absence of synaptotagmin I. Are these two release components differentially affected by the presynaptic changes of LTD, or is the mechanism of plasticity common to both? We find that LTD is expressed as parallel changes in the fast and slow components of release, and that this form of synaptic plasticity is still seen in the absence of functional synaptotagmin I. Any alterations in the presynaptic release machinery observed during LTD thus involve mechanisms shared by both modes of release.

Actin-dependent regulation of neurotransmitter release at central synapses.

Depolymerization of actin by latrunculin A transiently promotes neurotransmitter release. The mean rate of mEPSCs increases by a Ca2+-independent process, without a concomitant change in the mean amplitude. The readily releasable vesicle pool size and the rate of refilling of the readily releasable pool remain unaltered by latrunculin treatment. Evoked neurotransmitter release also increases in a manner consistent with an increase in vesicle release probability. The observed enhancement of neurotransmitter release is specific to actin depolymerization mediated by latrunculin A and is not caused by cytochalasin D. Our findings indicate that actin participates in a regulatory mechanism that restrains fusion of synaptic vesicles at the active zone.

SV2A and SV2B function as redundant Ca2+ regulators in neurotransmitter release.

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor isoform (SV2C) that resemble transporter proteins. We now show that SV2B knockout mice are phenotypically normal while SV2A- and SV2A/SV2B double knockout mice exhibit severe seizures and die postnatally. In electrophysiological recordings from cultured hippocampal neurons, SV2A- or SV2B-deficient cells exhibited no detectable abnormalities. Neurons lacking both SV2 isoforms, however, experienced sustained increases in Ca2+-dependent synaptic transmission when two or more action potentials were triggered in succession. These increases could be reversed by EGTA-AM. Our data suggest that without SV2 proteins, presynaptic Ca2+ accumulation during consecutive action potentials causes abnormal increases in neurotransmitter release that destabilize synaptic circuits and induce epilepsy.

Memory mechanisms. Photographic memory in flies.

A new study of olfactory memory formation in Drosophila shows that a delicate balance of CREB transcription factor activity may play a decisive role in triggering long-term memory.

Memory mechanisms. A common cascade for long-term memory.

Genetic, behavioral and electrophysiological approaches are beginning to unravel the mechanism of memory; cAMP-mediated gene expression appears to be universally required for establishing long-term memory.

Long-term potentiation. In pursuit of a retrograde messenger.

Platelet-activating factor is the latest candidate retrograde messenger involved in transmitting a signal from the postsynaptic to the presynaptic cell during long-term potentiation.

Memory mechanisms: the nociceptin connection.

A link between long-term potentiation (LTP) and memory has been supported by learning defects following treatments that impair LTP. Important new evidence for the link comes from mutant mice lacking the nociceptin receptor, which show improved learning and memory accompanied by enhanced LTP.

Synaptic plasticity: the basis of particular types of learning.

A new twist to the relationship between synaptic plasticity and learning and memory is revealed by the latest series of studies comparing animal behavior with electrophysiological recordings in the hippocampus.

The use of enzyme-linked immunosorbent assays (ELISA) for the determination of pollutants in environmental and industrial wastes.

Twelve enzyme-linked immunosorbent assays (ELISA), for the determination of surfactants [linear alkylbenzene sulfonates (LAS), alkyl ethoxylates (AE), and alkylphenol ethoxylates (APE)], endocrine disruptors [alkylphenol (AP), AP + APE, and bisphenol A (BPA)], estrogens [17beta-estradiol (E2), estrone (El), estrogen (ES: El + E2 + estriol (E3)), 1 7alfa-ethynylestradiol (EE2)], dioxins and polychlorinated biphenyls (PCBs), were validated on environmental water and industrial wastes. The lowest quantification limits of these ELISAs were 0.05 microg/L (BPA, E2, El, ES and EE2), 2 microg/L (AE), 3 microg/L (dioxins and PCBs), 5 microg/L (AP, AP + APE) and 20 microg/L (LAS and APE). To apply these ELISAs to environmental or industrial waste samples, simple and appropriate pre-treatment methods were also developed for each ELISA. With optimized pre-treatments, the values of ELISAs were well co-related, in all cases, to those of instrumental analytical methods such as liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and high-resolution gas chromatography mass spectrometry (HR-GC-MS), etc.

Participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain.

The participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain was examined. A reaction mixture of [1-14C]hexadecanoic acid with brain microsomes and NADPH formed two radioactive products having the same mobilities as pure hexadecanal (RF 0.61) and hexadecanol (RF 0.22), respectively, on TLC plates. The product of the RF 0.61 spot was further identified as hexadecanal using gas-liquid chromatography after methylation and TLC of its reduced product with LiAlH4 and semicarbazide. The ratio of hexadecanal to hexadecanol varied from 0.4 to 1.2 under the present experimental conditions. When solubilized rat brain microsomes were applied to a Sepharose 4B column coupled with the rabbit antibody raised against rat liver microsomal NADPH-cytochrome-c reductase, which reacts with aldehyde reductase from rat brain, the eluted fraction ceased to form [14C]hexadecanol but continued to form [14C]hexadecanal from [14C]hexadecanoic acid. These results strongly indicate that hexadecanal is the intermediate in the synthesis of hexadecanol from hexadecanoic acid in rat brain microsomes with the participation of microsomal aldehyde reductase.

Simultaneous determination of nineteen hallucinogenic tryptamines/beta-calbolines and phenethylamines using gas chromatography-mass spectrometry and liquid chromatography-electrospray ionisation-mass spectrometry.

To investigate the trend of non-controlled drugs of abuse, simultaneous analytical methods were developed using GC-MS and LC-ESI-MS for 8 tryptamines/beta-carbolines, 6 phenethylamines of typically non-controlled substances in Japan, and, additionally, five legally controlled tryptamines and phenethylamines originally found in fungi or plants. Moreover, the proposed methods were applied to analyses of these drugs in 99 kinds of products (a total number of 123 products purchased at adult shops or via the Internet over the past 2 years in Japan), which potentially advertised psychotropic/psychoactive effects. The samples were extracted with methanol under ultrasonication. After centrifugation, the extracts were filtered prior to injections. GC-MS analysis was performed using a DB-5MS capillary column. Regarding the LC-ESI-MS analysis; the separation of the target drugs was optimized on an ODS column in acetonitrile/MeOH (7:3)-10 mM ammonium formate buffer (pH 3.5)/acetonitrile (95:5) by a linear gradient program and a quantitative analysis was carried out by the monitoring of each [M+H]+ in the positive ion mode of ESI-MS. As a result of the analyses using GC-MS and LC-ESI-MS, 5-MeO-DIPT (the synthetic substance known by the street name "Foxy") was found in 8 out of the 99 kinds of products. Additionally, AMT (from brown powder), DMT (from dried plant), harmine and harmaline (from dried plant) were also found in some of the 99 products. These analytical methods could be useful for the investigation of the distribution of the non-controlled psychotropic tryptamines/beta-carbolines and phenethylamines in the market.

Induction of active systemic anaphylaxis by oral sensitization with ovalbumin in mast-cell-deficient mice.

Mast-cell-deficient W/W(v) mice were sensitized by oral administration of 0.1 and 1.0 mg ovalbumin (OVA) by gavage every day for 9 weeks, and active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of OVA. The production of OVA-specific IgE and IgG1 by oral immunization of the W/W(v) mice was high, and the production of IL-4 by splenocytes re-stimulated with OVA in vitro was increased. In contrast, production of OVA-specific IgG2a and IgG2b was low, and production of IFN-gamma by splenocytes after re-stimulation with OVA in vitro was rather decreased. These findings suggest that Th2-dominant helper T-cell activation had occurred. No increase in serum histamine level was observed following ASA induction. However, the plasma platelet-activating factor (PAF) levels of the mice sensitized with 0.1 and 1.0 mg OVA by gavage increased significantly. The increases in plasma PAF correlated well with the ASA-associated decreases in body temperature, suggesting that PAF plays an important role in ASA in W/W(v) mice. Taken together the above findings indicate that W/W(v) mice are a good model not only for studying induction of food allergy but also for examining the role of PAF in food-induced hypersensitivity.

Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice.

We studied the conditions needed to sensitize animals to the oral feeding of food allergens, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Brown Norway (BN) rats were sensitized by daily OVA (ovalbumin)-gavage or by drinking OVA containing water ad libitum and the ASA (active systemic anaphylaxis) response, as the immediate hypersensitivity response to antigen stimulation after oral sensitization, was examined. The oral administration of OVA by gavage produced a higher OVA-specific IgE response and an increase in serum histamine after antigen challenge, as compared to those produced by drinking water. Next, we examined the effect of murine age, the oral feeding technique and the oral feeding dose on sensitization using BALB/c, B10A and ASK mice. Twenty-week-old mice showed the strongest OVA-specific IgE and IgG1 responses and ASA-associated serum histamine contents increased with gavage in the three different age groups of BALB/c mice. Administering 0.1 mg of OVA by gavage daily for 9 weeks appeared to induce a higher response than administering 1 mg of OVA, in terms of OVA-specific IgE and IgG1 antibody responses and ASA responses. Among the three strains of mice, B10A mice exhibited the highest response in terms of OVA-specific IgE and IgG1 antibody and ASA responses. These findings suggested BN rats and B10A mice were suitable models for oral sensitization with antigen protein and that oral sensitization in mice requires low dose, intermittent antigen intakes.

Characterization of microsomal NADPH-dependent aldehyde reductase from rat brain.

An NADPH-dependent aldehyde reductase was purified from rat brain microsomes to electrophoretic homogeneity. The purified enzyme had a molecular weight of 75,000 and reduced long chain fatty aldehydes such as octanal and hexadecanal with higher affinity (Km values of 0.21 mM and 0.03 mM, respectively) than for various artificial carbonyl compounds such as p-nitrobenzaldehyde and p-nitroacetophenone (Km values of 0.31 mM and 1.4 mM, respectively). The purified microsomal aldehyde reductase also showed NADPH-cytochrome c reductase activity, and it could not be distinguished from NADPH-cytochrome c reductase in molecular weight (75,000), chromatographic behavior, electrophoretic mobility, or immunological properties. The solubilized microsomal fraction treated with steapsin lost the reductase activity for hexadecanal but not that for cytochrome c. These results suggest that the aldehyde reductase in brain microsomes is identical to NADPH-cytochrome c reductase and that a hydrophobic portion of the NADPH-cytochrome c reductase is required for the reduction of hexadecanal.