Embryonic mosaic deletion of APP results in displaced Reelin-expressing cells in the cerebral cortex.

It is widely accepted that amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease. In addition, APP has been proposed to have functions in numerous biological processes including neuronal proliferation, differentiation, migration, axon guidance, and neurite outgrowth, as well as in synapse formation and function. However, germline knockout of APP yields relatively subtle phenotypes, and brain development appears grossly normal. This is thought to be due in part to functional compensation by APP family members and other type I transmembrane proteins. Here, we have generated a conditional mouse knockout for APP that is controlled temporally using CreER and tamoxifen administration. We show that total cortical expression of APP is reduced following tamoxifen administration during embryonic time points critical for cortical lamination, and that this results in displacement of Reelin-positive cells below the cortical plate with a concurrent elevation in Reelin protein levels. These results support a role for APP in cortical lamination and demonstrate the utility of a conditional knockout approach in which APP can be deleted with temporal control in vivo. This new tool should be useful for many different applications in the study of APP function across the mammalian life span.

Thirty-second sampling of plasma growth hormone in man: correlation with sleep stages.

Both fasting and sleep increase the secretion of human GH and, therefore, might explain its predominantly nocturnal release. To study the precise temporal relationship between GH secretory episodes and cortical activity, GH measurements and electroencephalogram sleep stage recordings were performed every 30 s for 8 h in six young male volunteers fasted for 24 h. GH was measured in two drops of whole blood, which were directly sampled into the assay tube using a continuous blood withdrawal pump and a fraction collector. Concomitant serum sampling during a GH-releasing hormone test (n = 4) revealed a high correlation (r = 0.98) between GH measurements in serum and whole blood. GH pulses were objectively identified with Cluster analysis, and GH secretion rates were calculated with a waveform-independent deconvolution algorithm. When data were analyzed as replicates with 1-min intervals, the nocturnal pulse frequency was 1.2 pulses/h. Elimination of data points demonstrated 43% and 64% reductions in the number of GH pulses detected for 5- and 20-min sampling intervals, respectively. Mean GH concentrations and secretory rates were significantly higher during stage 3 and 4 sleep compared to stage 1, 2, and rapid eye movement sleep. GH secretory rates and peripheral GH concentrations were maximally correlated with sleep stage, with lags of 4.5 and 16 min, respectively, suggesting that maximal GH release occurs within minutes of the onset of stage 3 or 4 sleep. This temporal coincidence between pituitary GH secretion and delta sleep is consistent with cortical control over hypothalamic-pituitary function.

Characterisation of Ca2+ transport activity by white adipose tissue mitochondria.

Ca2+ transport in mitochondria isolated from rat white adipocytes has been examined and many of the properties found to be similar to those reported for mitochondria isolated from rat liver. Ca2+ transport is ruthenium red-sensitive (Ki approximately 5 pmol . mg protein-1), the affinity for free Ca2+ is high (Km approximately 3.3 microM) and the Vmax is 135 nmol Ca2+ . min-1 . mg protein-1 at 4 degrees C with 0.2 mM Pi present. Ca2+ transport is stimulated by increasing the medium [Pi], and is inhibited when ATP or Mg2+ is added to the incubation system and in contrast to brown adipocyte mitochondria, Ca2+ efflux is not promoted by Na+. White adipocyte mitochondria may play a rôle in the regulation of total cell calcium in this tissue.

Effect of depolarizing concentrations of potassium on calcium uptake and metabolism in rat liver.

Exposure of perfused livers of fed rats to 60 mM K+ induces rapid responses in the Ca2+-sensitive metabolic events, glycogenolysis, cytoplasmic and mitochondrial NADH/NAD ratios and octanoate oxidation. All increase within 45 s of K+ addition. Metabolic responses were not observed following K+ addition to livers perfused in the absence of added Ca2+. Movements of Ca2+ into the liver were suggested from experiments in which 45Ca2+ uptake was measured. The Ca2+ antagonists verapamil, diltiazem and Ni2+ essentially abolished changes to tissue metabolism and Ca2+ fluxes induced by K+ addition. K+-induced changes were consistent with Ca2+ channel activation.

Role of mucosal IgA in the resistance to Acanthamoeba keratitis.

PURPOSE: To determine whether oral immunization with Acanthamoeba castellanii antigens elicits mucosal antibodies of the IgA isotype and whether mucosal antibodies affect parasite adhesion to the corneal epithelium. METHODS: Chinese hamsters were immunized with 100 microg aqueous Acanthamoeba antigen mixed with cholera toxin (Ac-CT) and subsequently challenged with parasite-laden contact lenses that were applied to abraded corneal surfaces. Tears and stool samples were examined for the presence of Acanthamoeba-specific IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The effect of mucosal antibody on trophozoite binding to corneal epithelium and viability of trophozoites was examined in vitro. RESULTS: Hamsters immunized orally with Ac-CT showed significantly lower infection rates than did control groups (21.4% versus 72.6%). ELISA analysis of mucosal specimens showed the presence of parasite-specific IgA in stool samples and tears from hamsters orally immunized with Ac-CT, but not in control animals. In vitro assays showed that anti-Acanthamoeba IgA did not affect parasite viability. However, mucosal anti-Acanthamoeba IgA profoundly inhibited (>75%) the binding of parasites to corneal epithelial cells in vitro. CONCLUSIONS: Oral immunization with Ac-CT induces the production of parasite-specific IgA in mucosal secretions and prevents corneal infection. Mucosal antibody does not affect the viability of Acanthamoeba trophozoites but seems to prevent infection by inhibiting parasite binding to the corneal epithelium.

The role of macrophages in Acanthamoeba keratitis.

PURPOSE: Macrophages are thought to be the first line of defense in many infectious diseases and are present in high numbers in corneas with Acanthamoeba keratitis. Conjunctival macrophage depletion was performed in an animal model of Acanthamoeba infection to determine the importance of macrophages in this disease. METHODS: Selective elimination of macrophages was achieved by repeated subconjunctival injection of liposomes containing dichloromethylene diphosphonate in a Chinese hamster model of Acanthamoeba keratitis. RESULTS: Macrophage depletion affected the incidence, severity, and chronicity of keratitis. The incidence of infection in normal animals was approximately 60% but rose to 100% on day 4 in animals treated with liposomes containing dichloromethylene diphosphonate (C12MDP-LIP). Moreover, the clinical appearance of the keratitis in the C12MDP-LIP group was much more severe than in animals treated with liposomes containing phosphate-buffered saline at all time points. There was also a major change in the chronicity of keratitis, with an earlier onset and a prolonged and chronic course in the C12MDP-LIP treated hamsters. CONCLUSIONS: The profound exacerbation of Acanthamoeba keratitis in hamsters treated with C12MDP-LIP strongly suggests that macrophages play an important role in corneal infection with Acanthamoeba trophozoites, probably by acting as a first line of defense and eliminating significant numbers of Acanthamoeba trophozoites.

Schizophrenia, rheumatoid arthritis and trytophan metabolism.

Rheumatoid arthritis and schizophrenia have been described in early surveys as mutually exclusive disorders. Such claims are seen as especially interesting in view of: (1) indications that both illnesses often follow prodromes of severe psychological stress, (2) theories regarding hypermethylation of indoleamines producing endogenous psychotogens in schizophrenia, and (3) studies of rheumatoid arthritis reporting excessive binding of L-tryptophan to plasma protein, abnormalities of urinary tryptophan metabolites, decreased serotonin binding capacity of thrombocytes, and decreased MAO activity in joint fluid. Further comparative studies of tryptophan metabolism in schizophrenia and rheumatoid arthritis might enhance knowledge of pathogenesis in either or both diseases.

Sequential flow cytometry and fluorescence in situ hybridization for the study of formalin-fixed, paraffin-embedded breast cancer cells.

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.

In vitro characterization of Acanthamoeba castellanii cytopathic effect.

This study examined the mechanism of the cytopathic effect (CPE) of Acanthamoeba castellanii on human target cells. Pathogenic Acanthamoeba trophozoites were incubated with human ocular melanoma (OCM1) cells for 30 min, 1 hr, and 3 hr. The amoebae were treated with a calcium ionophore (A23187), phorbol myristate ester (PMA), calcium channel blocker (Bepridil), cytochalasin D, and L-leucyl-L-leucine methyl ester (leu-leu-OMe). Cytolysis was quantified using a spectrophotometric assay. Cocultures of amoeba and cells were also observed by transmission electron microscopy at 1, 2, and 3 hr. Results show that trophozoites formed pseudopodia that made intimate contact with the target cell membrane. Neither amebostomes nor phagocytosis was seen. The calcium ionophore A23187 increased the cytopathic effect of the trophozoites on the cultured OCM1. In contrast, cytochalasin D, Bepridil, and PMA reduced the cytopathic effect. Leu-leu-OMe did not result in killing of Acanthamoeba trophozoites. The results suggest that the cytopathic effect of Acanthamoeba trophozoites involves calcium channels and cytoskeletal elements. There was no evidence of trogocytosis or phagocytosis as sometimes occurs in cytolysis by other free-living amoeba. Although Acanthamoeba-mediated CPE in some ways resembles CPE produced by cytotoxic lymphocytes, the mechanisms are not identical.