Inhibition of p75 neurotrophin receptor does not rescue cognitive impairment in adulthood after isoflurane exposure in neonatal mice.

BACKGROUND: Isoflurane is widely used for anaesthesia in humans. Isoflurane exposure of rodents prior to post-natal day 7 (PND7) leads to widespread neurodegeneration in laboratory animals. Previous data from our laboratory suggest an attenuation of apoptosis with the p75 neurotrophin receptor (p75NTR) inhibitor TAT-Pep5. We hypothesized that isoflurane toxicity leads to behavioural and cognitive abnormalities and can be rescued with pre-anaesthesia administration of TAT-Pep5. METHODS: Neonatal mouse pups were pretreated with either TAT-Pep5 (25 µl, 10 µM i.p.) or a scrambled control peptide (TAT-ctrl; 25 µl, 10 µM i.p.) prior to isoflurane exposure (1.4%; 4 h) or control ( n = 15-26/group). Three to 5 months after exposure, behavioural testing and endpoint assays [brain volume (stereology) and immunoblotting] were performed. RESULTS: No significant difference was observed in open field, T-maze, balance beam or wire-hanging testing. The Barnes maze revealed a significant effect of isoflurane ( P = 0.019) in errors to find the escape tunnel during the day 5 probe trial, a finding indicative of impaired short-term spatial memory. No difference was found for brain volumes or protein expression. TAT-Pep5 treatment did not reverse the effects of isoflurane on neurocognitive behaviour. CONCLUSION: A single isoflurane exposure to early post-natal mice caused a hippocampal-dependent memory deficit that was not prevented by pre-administration of TAT-Pep5, although TAT-Pep5, an inhibitor of p75NTR, has been shown to reduce isoflurane-induced apoptosis. These findings suggest that neuronal apoptosis is not requisite for the development of cognitive deficits in the adults attendant with neonatal anaesthetic exposure.

Caveolae in ventricular myocytes are required for stretch-dependent conduction slowing.

Mechanical stretch of cardiac muscle modulates action potential propagation velocity, causing potentially arrhythmogenic conduction slowing. The mechanisms by which stretch alters cardiac conduction remain unknown, but previous studies suggest that stretch can affect the conformation of caveolae in myocytes and other cell types. We tested the hypothesis that slowing of action potential conduction due to cardiac myocyte stretch is dependent on caveolae. Cardiac action potential propagation velocities, measured by optical mapping in isolated mouse hearts and in micropatterned mouse cardiomyocyte cultures, decreased reversibly with volume loading or stretch, respectively (by 19±5% and 26±4%). Stretch-dependent conduction slowing was not altered by stretch-activated channel blockade with gadolinium or by GsMTx-4 peptide, but was inhibited when caveolae were disrupted via genetic deletion of caveolin-3 (Cav3 KO) or membrane cholesterol depletion by methyl-ß-cyclodextrin. In wild-type mouse hearts, stretch coincided with recruitment of caveolae to the sarcolemma, as observed by electron microscopy. In myocytes from wild-type but not Cav3 KO mice, stretch significantly increased cell membrane capacitance (by 98±64%), electrical time constant (by 285±149%), and lipid recruitment to the bilayer (by 84±39%). Recruitment of caveolae to the sarcolemma during physiologic cardiomyocyte stretch slows ventricular action potential propagation by increasing cell membrane capacitance.

Development of cataractous macrophthalmia in mice expressing an active MEK1 in the lens.

PURPOSE: To characterize the extracellular signal-regulated kinase (ERK) pathway in the lens and to try to understand how this pathway contributes to lens function and cataractogenesis. METHODS: The members of the ERK pathway in the lens were examined by Western blotting, immunohistochemical staining, and kinase assay. A gain-of-function approach was used to perturb the ERK pathway in the lenses of transgenic mice via expression of a constitutively active mutant of the mitogen-activated protein kinase kinase 1 (MEK1(E)), the direct upstream kinase of the ERK1 and ERK2 kinases, under the alphaA-crystallin promoter. RESULTS: The presence of an active ERK pathway was found in lens epithelial cells and in differentiating fibers. Transgenic mice that expressed MEK1(E) developed postnatal cataracts as well as macrophthalmia. Distinct morphologic alterations, such as lens enlargement, swelling fiber cells, enlarged extracellular space, and vacuole formation, were observed in the lenses of these transgenic mice. A significant increase in the glucose transporter 1 (GLUT1) level, as well as in the glucose level, was detected in the lens. CONCLUSIONS: The MAP kinase pathway is involved in the regulation of glucose metabolism and balance in the mouse lens. Moreover, the alteration of MAP kinase activity in the lens is sufficient to cause cataract formation with enlarged extracellular space and vacuoles in the differentiating fibers. This transgenic mouse may provide a useful model for understanding the mechanism(s) for some aspects of human cataracts.

Heterogeneity of astroglia cultured from adult human temporal lobe.

This study characterized the morphological and electrophysiological diversity of astroglia cultured from adult human cerebral temporal lobe, and explored the influence of the cytokine interleukin-1beta on these cells. The cultures contained astroglia positive for glial fibrillary acidic protein which were flat, bipolar or multipolar in shape and variable in size. A subpopulation of the bipolar and multipolar cells was positive for S100 protein. The most striking feature of these cultures was the presence of glia with long (600 micrometer) processes with few branches or only terminal branches. Patch clamp recordings of the non-stellate process bearing cells revealed prominent inward Na(+) and transient and sustained outward K(+) conductances. Distinct differences in the relative proportion of these conductances were evident among cells but did not appear to be correlated with cell morphology. Treatment of cultures with interleukin-1beta for 96 h did not change total protein content, but increased the content of S100beta protein and decreased the content of glial fibrillary acidic protein. The findings indicate that cultures of adult human cerebrum contain subpopulations of morphologically and electrophysiologically pleomorphic glial fibrillary acidic protein positive astroglia, exhibit increased levels of the neurotrophic factor S100beta when exposed to interleukin-1beta, and may serve as a useful model for investigation of glial involvement in neuropathology.

Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways.

Toxoplasma gondii actively penetrates its vertebrate host cell to establish a nonfusigenic compartment called the parasitophorous vacuole (PV) that has previously been characterized primarily in phagocytic cells. To determine the fate of this unique compartment in nonphagocytic cells, we examined the trafficking of host cell proteins and lipids in Toxoplasma-infected fibroblasts using quantitative immunofluorescence and immunoelectron microscopy. Toxoplasma-containing vacuoles remained segregated from all levels of the endocytic pathway, as shown by the absence of delivery of transferrin receptors, mannose phosphate receptors, and the lysosomal-associated protein LAMP1 to the vacuole. The PV was also inaccessible to lipids (DiIC16, and GM1) that were internalized from the plasma membrane via the endocytic system. In contrast, vacuoles containing dead parasites or zymosan sequentially acquired both endosomal and lysosomal protein markers and host lipids, reflecting the competency of fibroblasts to process phagocytic vacuoles. The mature PV often lies adjacent to the host cell Golgi, suggesting that it may intersect with vesicles from the exocytic pathway. Despite this proximity, the PV was inaccessible to nitrobenzadiazole-labeled sphingolipids exported from the Golgi and did not contain the host protein markers AP1 or beta-COP. Our results demonstrate that Toxoplasma resides in a compartment that excludes delivery of protein and lipid components from the host cell endocytic and exocytic pathways.

Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1 and exists primarily in a globular form.

Actin is a highly conserved microfilament protein that plays an important role in the invasion of host cells by the protozoan parasite Toxoplasma gondii. We have characterized the ACT1 gene and localized the conventional isoform of actin that it encodes within T. gondii. The predicted amino acid sequence of ACT1 was most similar to two other parasite actins, Plasmodium falciparum Pfact-1 (93.1% identical) and Cryptosporidium parvum actin (88.1%): among vertebrate actins, ACT1 was most closely related to the mammalian beta and gamma (83%) actin isoforms. Actin-specific antibodies and fluorescently labeled DNAse I were used to localize actin in T. gondii tachyzoites by immunofluorescence and immunoelectron microscopy. Actin was detected beneath the parasite cell membrane and in clusters scattered within the cytosol of T. gondii tachyzoites. Actin filaments were not detected in detergent-solubilized parasites separated by high speed centrifugation, indicating that actin exists primarily in a globular form in T. gondii.

Activated macrophage/microglial cells can promote the regeneration of sensory axons into the injured spinal cord.

A prominent role for phagocytic cells in the regenerative response to CNS or PNS injury has been suggested by numerous studies. In the present work we tested whether increasing the presence of phagocytic cells at a spinal cord injury site could enhance the regeneration of sensory axons from cut dorsal roots. Nitrocellulose membranes treated with TGF-beta or coated with microglial cells were cotransplanted with fetal spinal cord tissue into an injured adult rat spinal cord. Cut dorsal roots were apposed to both sides of the nitrocellulose. Four weeks later, animals were sacrificed and spinal cord tissue sections were processed for immunocytochemical detection of calcitonin gene-related peptide (CGRP-ir) to identify regenerated sensory axons. Adjacent sections were processed with the antibody ED-1 or the lectin GSA-B4 for detection of macrophage/microglial cells in association with the regrowing axons. Qualitative and quantitative data indicate a correlation between the pattern and extent of axonal regeneration and the presence of phagocytic cells along the nitrocellulose implant. Axonal regeneration could be experimentally limited by implanting a nitrocellulose strip treated with macrophage inhibitory factor. These results indicate that increasing the presence of activated macrophage/microglial cells at a spinal cord injury site can provide an environment beneficial to the promotion of regeneration of sensory axons, possibly by the release of cytokines and interaction with other nonneuronal cells in the immediate vicinity.

Stable DNA transformation of Toxoplasma gondii using phleomycin selection.

Toxoplasma gondii (Tg) is an obligate intracellular protozoan parasite that is an important opportunistic pathogen in humans. To provide additional tools for molecular genetic analysis, we have developed a set of vectors for DNA transformation in Tg based on selection with the antibiotic phleomycin (Ph). These new vectors rely on the flanking sequences from the parasite genes GRA1, GRA2 or SAG1 to drive expression of the Tn5 ble gene encoding resistance to the DNA intercalating drug Ph (PhR). Treatment of extracellular parasites was used to select stable PhR transformants by plaque formation on host cell monolayers. Transfection of linear or circular forms of the pGRA1/ble, pGRA2/ble or pSAG1/ble vectors by electroporation resulted in stable transformation with an efficiency of approx. 10(-4)/micrograms DNA. Stable transformants contained 1-5 copies of ble that were integrated at non-homologous sites in the parasite nuclear genome. Ble provides a new dominant selectable marker for safe, efficient and rapid isolation of stable DNA transformants in Tg.

Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that actively invades virtually all types of nucleated cells, surviving within a specialized vacuole called the parasitophorous vacuole. Shortly after invasion, the parasite modifies this vacuole by secreting a variety of proteins from electron-dense storage granules. Additionally, the parasite forms a network of membranous tubules within the lumen of the vacuole and connecting with the vacuolar membrane. We have used immunolabeling and cell fractionation to examine the secretion of two dense granule proteins, GRA1 and GRA2, which are involved in formation of the intravacuolar network. Following host-cell invasion, GRA1 was secreted into the lumen of the vacuole as a soluble protein that subsequently became peripherally associated with the network. In addition to being secreted as a soluble protein from dense granules, GRA2 was secreted within multi-lamellar vesicles released from a specialized posterior invagination of the parasite. The multi-lamellar vesicles assemble to form the intravacuolar network, which contains an integral membrane form of GRA2. These findings indicate that Toxoplasma has a highly developed regulated exocytosis pathway that modifies the parasitophorous vacuole by secretion of soluble proteins and by a novel process of membrane secretion.

Toxoplasma gondii: secretion of a potent nucleoside triphosphate hydrolase into the parasitophorous vacuole.

Toxoplasma gondii is an obligate intracellular parasite capable of invading a wide range of host cells where it residues in a specially modified compartment termed the parasitophorous vacuole. This compartment provides a protected environment for the parasite which enters a rapid growth phase shortly after invasion. To identify functional components of this interface, we have used immunolabeling and cell fractionation to localize the potent nucleoside triphosphate hydrolase (NTPase) produced by Toxoplasma. In extracellular tachyzoites, NTPase was not exposed on the cell surface but was localized in small vesicles, called dense granules, scattered throughout the cell cytoplasm. Shortly following invasion. NTPase was secreted from dense granules and occupied the lumen of the parasitophorous vacuole where it was often associated with the intravacuolar network. NTPase was exclusively found in the supernatant in cell fractionation studies of both extracellular tachyzoites lysed by freeze-thaw and following secretion into the parasitophorous vacuole. These studies provide evidence for the rapid secretion of NTPase into the parasitophorous vacuole, where it may play a key role in processing of nucleotides for purine salvage by the parasite.

Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy.

The protozoan parasite Toxoplasma gondii infects a wide range of vertebrate hosts and is an important opportunistic pathogen in immunocompromised humans. Although Toxoplasma is amenable to both biochemical and cellular experimental approaches, the molecular basis of its success as an intracellular parasite is poorly understood. To provide a system for molecular genetic analyses, we have developed a stable DNA transformation system for Toxoplasma based on complementation of its naturally occurring tryptophan auxotrophy. Complementation was accomplished by expressing the Escherichia coli trpB gene, encoding the beta subunit of tryptophan synthase (EC 4.2.1.20), the enzyme that catalyzes the formation of tryptophan from indole plus serine. Transformants were obtained by electroporation of a plasmid, called SAG1/trpB, containing the trpB gene flanked by Toxoplasma surface antigen 1 (SAG1) gene sequences and selection for growth on indole. Transformants were obtained with circular forms of the SAG1/trpB plasmid with efficiencies of 10(-4) per cell. Transformation with either circular or linear SAG1/trpB resulted in integration into the genome at distinct, nonhomologous sites. Trp+ transformants typically contained tandemly repeated copies of the SAG1/trpB plasmid and were stable in the absence of continued selection. The Trp phenotype provides a dominant selectable marker that should allow expression of foreign or altered genes in Toxoplasma and facilitate molecular analyses of genes important for intracellular survival.

Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue.

The H(+)/ATP stoichiometry was determined for the plasma membrane H(+)-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H(+)/ATP stoichiometry utilized a kinetic approach where rates of H(+) influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H(+) influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H(+) leakage from a steadystate pH gradient after stopping the H(+)-ATPase or utilizing a mathematical model which describes the net transport of H(+) at any given point in time. When the rate of H(+) influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H(+)/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (DeltaG(atp)), this value for H(+)/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.

Change in Target Molecular Size of the Red Beet Plasma Membrane ATPase during Solubilization and Reconstitution.

The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of gamma-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity.

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum.

Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a primary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca(2+), A23187 stimulated-ATPase). Both calcium uptake and Ca(2+), A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [gamma-(32)P]-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization.