Rate and Duration of Stimulation Determine Presynaptic Effects of Haloperidol on Dopaminergic Neurons

Abstract: Superfused rabbit neostriatal slices prelabeled with [³H]dopamine ([³H]DA) were depolarized with electrical pulses (12 V, 1 ms). Although transmitter release showed a proportional increase with a greater number of pulses (30-360 pulses), flat frequency-release curves were obtained. Haloperidol (0.03–0.3 μ m ) enhanced ³H overflow without affecting its metabolism or time course, and antagonized apomorphine-induced inhibition of transmitter release. Maximal enhancement of release by haloperidol was obtained with 30–60 pulses delivered at a rate of 3 Hz, whereas much less facilitation of release was seen at 0.3 and 1 Hz (30–90 pulses) or with 360 pulses at either of the three frequencies. Therefore, the slope of the frequency-release curve was markedly increased by haloperidol. These results indicate that activation of presynaptic DA receptors, and thus facilitation of release by haloperidol was highly dependent on the rate and duration of stimulation of striatal dopaminergic terminals. In these neurons the feedback loop seems to act physiologically to depress the slope of the frequency-release curve.     

Chemiluminescence activity in whole blood phagocytes of dogs naturally infected with Leishmania infantum

Dogs are the domestic reservoir of Leishmania infantum, a vector-borne intracellular protozoan agent of human visceral leishmaniasis. The role of polymorphonuclear leukocytes (PMNs) in the immune defence against this parasite has been poorly studied. We have investigated the function of peripheral blood PMNs in naive beagle dogs that have been naturally exposed to phlebotomine vectors in an area highly endemic for canine leishmaniasis, and found infected by Leishmania at the end of the transmission season. Whole blood phagocyte oxidative metabolism was assessed by a rapid method that determines a luminol-amplified chemiluminescence (CL) emission. This was evaluated using either a soluble stimulant, phorbol mirystate acetate (PMA), or phagocytic stimuli, such as zymosan unopsonized (ZYM) or opsonized with autologous serum (OPZ). In blood samples taken 2 months after exposure to Leishmania transmission, data on CL emission revealed a significant decrease of reactive oxygen intermediates (ROI) production in the presence of both PMA and ZYM, compared with blood samples obtained from dogs before exposure. On the contrary, no variations in CL emission were detected in presence of OPZ. Our data indicate that immunological changes occur early in canine leishmaniasis and confirm that the role of PMNs and their products need to be clarified. Copyright © 2000 John Wiley & Sons, Ltd.     

Enhancing Effect of Manganese on L-DOPA-Induced Apoptosis in PC12 Cells

L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 microM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.     

Identification of Pediococcus acidilactici and Pediococcus pentosaceus based on 16S rRNA and ldhD gene-targeted multiplex PCR analysis

A multiplex PCR assay that can readily and unambiguously identify Pediococcus acidilactici and Pediococcus pentosaceus strains was developed to give an easy-to-read profile based on the amplification of a 16S rRNA gene fragment, specific for each species, and a d-lactate dehydrogenase gene fragment specific for Pediococcus acidilactici strains.     

Cloning of murine CDK9/PITALRE and its tissue-specific expression in development

The cdc2-family of serine/threonine kinases and their binding partners recently were implicated in developmental roles. We previously cloned a cdc2-related kinase, cdk9/PITALRE, that is able to phosphorylate the retinoblastoma protein in vitro. We describe here the cloning and the characterization of the mouse homolog of cdk9/PITALRE. The murine cDNA is 98% identical with humans and is expressed at high levels in brain and kidney tissues. The kinase activity and protein expression of cdk9/PITALRE were highest in terminally differentiated tissues such as the muscle and brain. In situ immunohistology and immunofluorescence detected cdk9/PITALRE protein not only within terminally differentiated cells such as muscle and neuronal cells, but also in proliferating cells. C2C12 and P19 cells induced to differentiate along muscle and neural lineages peaked in cdk9/PITALRE kinase activity at the end of differentiation. These results suggest that, among other roles, cdk9/PITALRE plays a role not unlike cdk5 in the differentiation of certain cell types. J. Cell. Physiol. 177:206–213, 1998. © 1998 Wiley-Liss, Inc.     

Rapid reversed-phase high-performance liquid chromatographic method with double derivatization for the assay of urinary hydroxyproline

An HPLC method with two derivatizations, the first with o-phthaldehyde in order to eliminate interferences due to some primary amino acids eluting with retention times similar to those of hydroxyproline and the second with dabsyl chloride, was developed and evaluated. Calibration graph linearity, influence of agitation and temperature on the preparation of the first derivative and the influence of the detection wavelength were assessed. The analysis time is shorter in comparison with other available methods, and therefore this method is suitable for laboratories that analyse both small and large series of samples.     

Cancer cells adapt FAM134B/BiP mediated ER-phagy to survive hypoxic stress

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.Subject terms: Cell biology, Cancer