A novel quantitative polymerase chain reaction to monitor urinary tract mycoplasma infection in a dog.

The aim of the study was to develop a quantitative real-time PCR assay for diagnosis and monitoring of mycoplasma urinary tract infections (UTI) in a dog. An English Cocker Spaniel dog with the history of urinary tract infection was physically examined and laboratory findings identified chronic renal insufficiency and urinary tract infection. Attempts to culture organisms from pyuric urine failed, and empirical antibiotic therapy did not resolve the pyuria. A mycoplasma species most closely resembling Ureaplasma canigenitalium was identified in urine samples by conventional PCR and sequencing. A quantitative PCR method was developed to monitor and finally verify successful treatment. This novel approach to monitoring mycoplasma urinary tract infections is conceptually simple, and provides rapid results. It may have wider application in monitoring treatment efficacy for infections with other Mycoplasma spp. as well as additional organisms that are difficult to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we highlight two different findings, detection of Ureaplasma canigenitalium in a dog with chronic urinary tract infection and development of a quantitative real-time PCR test to track treatment results in an infected dog. This report is the first report of detection of U. canigenitalium in one dog in Australia. This novel qPCR method for monitoring mycoplasma urinary tract infections is conceptually simple and provides results fast. It will have wider applications in monitoring treatment efficacy for infections with mycoplasmas and mycoplasma-like organisms that are difficult to culture, and provides a sensitive guide to treatment progress.

Characterization of a tropane radioligand, [(3)H]2beta-propanoyl-3beta-(4-tolyl) tropane ([(3)H]PTT), for dopamine transport sites in rat brain.

PTT (2beta-propanoyl-3beta-[4-tolyl] tropane) is a tropane analog relatively selective for dopamine transporters in binding and uptake assays in vitro, with long-acting psychostimulant properties in vivo. To explore its utility in binding to dopamine transporters, [(3)H]PTT was synthesized and assayed for binding in rat striatal membranes and by in vitro autoradiography. In membranes, binding of [(3)H]PTT was saturable to a single class of binding sites with a K(D) value of 3 nM. The pharmacology of [(3)H]PTT binding in striatal membranes was consistent with that of a ligand selective for dopamine transporters, with dopamine-selective compounds being significantly more potent in displacing [(3)H]PTT binding than those for 5-HT or norepinephrine transporters. Although the ability of various transporter inhibitors to displace both [(125)I]RTI-55 and [(3)H]PTT binding correlated significantly with each other, there was a better correlation of inhibitor potencies versus [(3)H]PTT binding and dopamine uptake than versus [(125)I]RTI-55 binding and dopamine uptake. The differences in correlations were most noticeable for compounds relatively selective at the 5-hydroxytryptamine (serotonin) transporter. The autoradiographic distribution of [(3)H]PTT binding in coronal sections was consistent with the known distribution of the dopamine transporter, with high levels of binding evident in caudate nucleus, nucleus accumbens, and olfactory tubercle. Moderate densities of [(3)H]PTT binding were also observed in substantia nigra pars compacta, and ventral tegmental area, as well as in the anterior cingulate cortex and portions of the hypothalamus. In addition, nonspecific binding was less than 5% of total binding. Thus, [(3)H]PTT provides an accurate and convenient marker for the dopamine transporter.

Astrocyte and neuron coculturing method.

It is customary to credit Rudolf Virchow (1) with the discovery of neuroglia (see ref. 2). As a practicing pathologist who was familiar with inflammatory processes in the brain, Virchow opposed his contemporaries' assertion that the brain was void of connective tissue. He hypothesized that underneath the single-cell layer of the ependyma, the ventricles were lined with connective tissue cells that were capable of mounting inflammatory responses, and referred to these cells as "Nervenkitt" or "nerve putty." Although erroneous, this coined term has persisted as the preferred generic term, or in its shortened form "glia," for a class of nonexcitable brain cells. Classification and histological characterization of the true nature of the various neuroglial types followed the development of impregnation techniques by Golgi and Ramon y Cajal in the late 1800s. By the 1920s, the major forms of glial cells had been recognized and identified. Their basic structures and relationships with other critical parts of the nervous system were also beginning to emerge.

Prolonged dopamine and serotonin transporter inhibition after exposure to tropanes.

Cocaine and tropane analogs are known to interact with biogenic monoamine transporters by inhibiting amine uptake. Previous in vivo studies have demonstrated that some of these tropanes produce a longer lasting behavioral effect compared with cocaine. We have previously examined several tropane analogs and found a difference in their relative affinities for dopamine (DA) and serotonin (5-HT) transporters. The purpose of this study was to determine the recovery time of transporter function in vitro and in vivo comparing cocaine with the tropane analogs WF-11 (PTT, selective for DA transporters), WF-31 (selective for 5-HT transporters) and WF-23 (highly potent at both DA and 5-HT transporters). In vitro, using primary rat brain cultures of either midbrain or raphe regions, the recovery of the ability to transport either [3H]dopamine or [3H]serotonin, respectively was evaluated at 0, 3, 24, 48, 120 and 240 h after a 1 h exposure to cocaine and tropane analogs. The tropanes exhibited clearance half-lives ranging from 12 to 69 h, while cocaine, on the other hand, exhibited a clearance half-life of approximately 6 h. In studies utilizing [125I]RTI-55 binding, intraperitoneal injections of cocaine and WF-23 into the rat resulted in striatal clearance half-lives ex vivo that were almost identical to those obtained in vitro. These data suggest that the tropanes bind to and reduce transporter function for prolonged periods of time (up to 10-fold longer than cocaine) and those compounds with the highest affinity may produce a pseudo-irreversible inhibition of transporter function.

Long-acting blockade of biogenic amine transporters in rat brain by administration of the potent novel tropane 2beta-propanoyl-3beta-(2-Naphthyl)-tropane.

2beta-Propanoyl-3beta-(2-naphthyl)-tropane (WF-23) is a potent cocaine analog with activity at dopamine and serotonin transporters. The purpose of these experiments was to characterize the time course of effects of acute administration of WF-23 on spontaneous locomotion and biogenic amine transporters. Rats received injections i.p. with WF-23 (1 mg/kg), cocaine (30 mg/kg) or vehicle and locomotor activity was measured at various times postinjection. Animals were killed immediately after behavioral activity. Locomotor activity was significantly increased by WF-23 administration, reaching maximum at 4 hr and persisting for 24 hr. Cocaine-elicited elevations in locomotor activity occurred only at the earliest times. WF-23 decreased DA transporter binding in striatal membranes ([125I]RTI-55 binding), with >50% loss in binding for up to 49 hr postinjection. WF-23 increased the Kd of the high affinity site, with no effect on Bmax. Cocaine depressed binding (20%) only at the earliest times. WF-23 decreased levels of [3H]WIN 35,428 binding sites up to 95% of control in both dorsal and ventral striatum with a similar time-course when assessed autoradiographically. WF-23 also reduced [3H]citalopram binding to serotonin transporter sites throughout the brain. The slow onset and very long duration of action of WF-23, taken together with its actions at dopamine and serotonin transporters, suggest a potential role for treatment of disorders characterized by their involvement of these neural systems.

Methamphetamine-induced alterations in dopamine transporter function.

Repeated methamphetamine (METH) administration has been shown to produce differing neurochemical as well as behavioral effects in rats. This study was designed to examine the effects of acute and chronic METH exposure on uptake and release of [3H]dopamine (DA) in cultured midbrain dopamine neurons to determine if persistent neuronal adaptations ensue. In addition, we have assessed DA D2 receptor function to determine if chronic METH alters this receptor. Fetal midbrain cultures were exposed to METH (1, 10 microM) for 5 days and dopaminergic function examined 1 or 7 days after drug removal. The ability of METH to release [3H]DA was compared to other releasing agents as well as several potent uptake inhibitors. Chronic exposure to a release-promoting concentration of METH resulted in either no change or a reduction in [3H]DA release upon subsequent METH challenge. Pretreatment with METH was also found to cause a decrease in the Bmax for [3H]raclopride binding, suggesting that persistently elevated DA levels cause a downregulation of DA D2 receptors. Examination of transporter kinetics utilizing initial velocity of uptake revealed that METH treatment caused a significant decrease in affinity (K(m)) for the substrate (DA), while not altering the maximal velocity of uptake (Vmax). Binding studies with [125I]RTI-55 revealed that there was no alteration in either the Bmax or Kd for this ligand, suggesting that the changes induced by METH treatment are due to alterations in K(m) and not in the number of DA transport sites. The results from these studies indicate that METH treatment produces a modification in transporter function which may be associated with both the altered uptake and release of [3H]DA. These changes have broad implications for the regulation of transporter activity not only because of the relevance to pre-synaptic mechanisms controlling neurotransmission, but also to the importance of the neuronal adaptation that occurs in response to chronic METH exposure.

Radiochemical neutron activation analysis for trace chlorine in steels and alloys.

Chlorine is usually present at low concentrations in reactor materials and thermal neutron activation of (35)Cl produces (36)Cl, which has a long half-life and is a radionuclide of significance in nuclear waste disposal. This paper describes a radiochemical method that has been developed to measure low concentrations of Cl in reactor stainless steels, so that the amount of (36)Cl in radioactive wastes can be estimated. The method is based on the irradiation of a 1 g sample in a thermal neutron flux of 10(16) n m(-)(2) s(-)(1), followed by dissolution in HNO(3) with the addition of stable KCl carrier/tracer. The Cl is precipitated as AgCl, and the recovery is measured gravimetrically. The (38)Cl, which has a half-life of 37 min, is measured with γ-ray spectrometry. The entire process, from irradiation to the end of counting, takes ∼1.5 h. The recovery is near-quantitative, and the detection limit for Cl in most stainless steels is below 1 mg/kg.

The determination of 129I in milk and vegetation using neutron activation analysis.

A new method has been developed to measure 129I in the environment with detection limits below 10 mBq/kg of vegetation and 10 mBq/l of cows' milk. The method is based on extraction of 129I from the milk or vegetation sample, onto an ion exchange resin. An inactive carrier of 127I is added to the sample before separation, to monitor losses throughout the entire procedure. The ion exchange resin is irradiated for 7.5 h in a neutron flux of 10(16) n m-2 s-1 to induce the 129I(n, gamma) 130I reaction with thermal neutrons. The 127I carrier undergoes a (n,2n) reaction with fast neutrons to produce 126I. Iodine is extracted from the ion exchange resin after irradiation with an elution scheme which removes contamination from the radionuclide 82Br, the main interference in the analysis. Finally iodine is precipitated as AgI for gamma ray analysis. The sample is counted for 3 h on a Ge semiconductor detector to measure the radionuclide 130I, which has a half life of 12.4 h and 126I, which has a half life of 13.0 days. The measured 130I activity is compared to a known standard to deduce the amount of 129I in the sample, and the concentrations are corrected for losses during processing using the measured activity of 126I. The detection limits for 129I by this method are below 10 mBq/l for milk samples and 10 mBq/kg for vegetation. In addition to routine monitoring of milk and grass samples the method has been used to measure 129I deposition on grass and soils in a field near the Sellafield plant. Results of these analyses, along with measurements of 129I in air and rainfall using the same methodology, have been used to determine deposition velocity and retention coefficients of 129I to grass.

HIV-1 gp120-induced neurotoxicity to midbrain dopamine cultures.

HIV-1-associated cognitive/motor dysfunction is a frequent neurological complication of acquired immunodeficiency syndrome (AIDS) and has been termed AIDS dementia complex (ADC). The HIV-1 envelope glycoprotein gp120 has been implicated in producing brain injury associated with ADC. The purpose of the present study was to determine if gp120-induced neurotoxicity is associated with damage to dopaminergic systems. Exposure of rat midbrain dopamine cultures to gp120 for 3 days reduced the ability of dopaminergic cells to transport this amine and also resulted in a reduction in dopamine neuron process length while it did not alter either dopamine cell number or the total number of neuronal cells. These detrimental effects of gp120 were prevented by an NMDA receptor antagonist (MK-801) or by preincubation with anti-gp120 antibody. These results suggest that dopaminergic neuronal damage may contribute to the manifestations of AIDS dementia complex.

Release of serotonin induced by 3,4-methylenedioxymethamphetamine (MDMA) and other substituted amphetamines in cultured fetal raphe neurons: further evidence for calcium-independent mechanisms of release.

The substituted amphetamines 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-chloro-amphetamine (PCA) and fenfluramine (FEN) all exert their effects by releasing serotonin (5-HT) from presynaptic nerve terminals. In the current study, we examined the ability of these agents to induce the release of 5-HT in culture fetal raphe neurons. The data indicate that the rank order of release potencies for these agents was (+/-)PCA>(+)MDMA=(+)MDA=(+/-)FEN. Studies examining the role fo calcium in 5-HT release demonstrate that preventing calcium influx with L- and N-type calcium channel blockers inhibits potassium-stimulated release of -3H-5-HT but has no effect on release induced by the substituted amphetamines. Furthermore, omitting calcium from the extracellular media or depleting the vesicular pool of neurotransmitter with continual potassium stimulation did not affect the release of -3H-5-HT induced by these compounds. Administration of fluoxetine prior to the substituted amphetamines significantly attenuated the releasing effects of these agents, while producing no effect on potassium-stimulated release. These results are consistent with the notion that the amphetamines induce release of cytoplasmic 5-HT via the plasma membrane transporter.

Novel 2-substituted cocaine analogs: uptake and ligand binding studies at dopamine, serotonin and norepinephrine transport sites in the rat brain.

A novel scheme utilizing vinylcarbenoid precursors has been developed for the synthesis of tropane analogs of cocaine. Specificities of these compounds for dopamine (DA), serotonin (5-HT) or norepinephrine (NE) transporters were determined by both uptake inhibition and binding assays. In each of the analogs, the aryl group at position 3 was bound directly to the tropane ring (as in WIN-35,428), and methyl or ethyl ketone moieties were present at position 2 in lieu of the ester linkage present in cocaine. The addition of methyl or ethyl ketone groups in position 2 did not affect potency compared to ester groups in the same position, but substituents on the benzene ring greatly affected potencies. The analogs could be categorized according to their relative specificity, which consisted of those selective for DA and NE transporters with little affinity for the 5-HT transporter (e.g., WF-39), those selective for only the DA transporter (e.g., WF-29) and those selective for the 5-HT transporter (e.g., WF-31) with much less affinity for the DA and NE transporters. There also were those analogs (e.g., WF-23) with high affinity, but with equal affinity at all three transporters. The analogs displayed significant correlation between uptake inhibition and binding displacement at DA, 5-HT and NE transport sites. These results suggest that large variations in the tropane structure do not result in a differentiation between binding at biogenic amine transporters and inhibition of amine uptake.

Similar properties of fetal and adult amine transporters in the rat brain.

A variety of drug classes, including psychomotor stimulants and antidepressants, interact with monoamine transporters in order to exert their effects. Although these transporters have been extensively characterized in the adult brain, little is known about uptake mechanisms in the fetal system. High affinity dopamine (DA) and serotonin (5-HT) uptake in the striatum and frontal cortex, respectively, were examined in rat fetuses (embryonic day 20; E-20). These results were then compared to uptake in adult rat synaptosomal preparations of the same regions. The data indicate that the fetal (E-20) uptake mechanism is sodium-dependent. Furthermore, the potency of various agents to inhibit transporter function was assessed. These drugs produced a concentration-dependent inhibition of uptake, and the resulting IC50 values were not significantly different from those obtained in the adult preparations. Our results provide evidence that the affinity of monoamine uptake inhibitors for fetal (E-20) DA and 5-HT transporters is similar to that observed with adult transporters. This observation has broad implications when considering neuronal development and in utero exposure to drugs that exert their effects through these transporters.

Nemesis revisited: tuberculosis infection in a New York City men's shelter.

In November 1990, a screening was conducted to determine the point prevalence of tuberculosis infection in a volunteer sample of homeless men (n = 161) living in a congregate shelter in New York City. Of those for whom we have results (n = 134), 79% were positive for tuberculosis. The mean length of shelter stay from date of shelter entry was 31.8 months and was significantly associated with the tuberculosis infection rate. The findings suggest that crowded living conditions and the presence of a stable resident pool in crowded congregate shelters may be associated with transmission of tuberculosis infection.

Differing neurotoxic potencies of methamphetamine, mazindol, and cocaine in mesencephalic cultures.

The potent reinforcing effects of methamphetamine and cocaine are thought to be mediated by their interactions with CNS dopamine neurons. Both stimulants share the ability to block dopamine uptake potently, and methamphetamine can release cytoplasmic dopamine as well. There is also abundant evidence demonstrating the neurotoxic effects of methamphetamine. There are, however, limited studies that attempt to discern the neurotoxic mechanisms of these agents. The purpose of the present study was to characterize and compare the chronic in vitro effects of methamphetamine, cocaine, and the dopamine uptake blocker, mazindol, on cultured fetal mesencephalic dopamine neurons. Our studies examined biochemical mechanisms to evaluate the contribution of reuptake blockade versus release of dopamine. Using a dispersed cell preparation of fetal mesencephalon, cultures were treated for 5 days with the three uptake blockers. Dopamine function was assessed by measuring high-affinity [3H]dopamine uptake and by examining cultures for the presence of tyrosine hydroxylase-immunopositive neurons. Nonspecific neurotoxicity was assessed by staining for neuron-specific enolase and measuring lactate dehydrogenase activity. The results indicate that repeated administration of high concentrations of methamphetamine (10(-4) and 10(-3) M) caused a generalized neurotoxicity whereas the effects of 10(-5) M methamphetamine appeared to be specific to dopamine cells. Likewise, treatment of the cultures with mazindol (10(-6) M) resulted in reduced dopamine uptake while not significantly affecting neuron-specific enolase or tyrosine hydroxylase immunostaining. On the other hand, repeated exposure to cocaine (10(-5) and 10(-4) M) did not alter dopaminergic function in these cultures. The different mechanisms of action of these stimulants may explain the differences in neurotoxic potency of these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term cocaine administration is not neurotoxic to cultured fetal mesencephalic dopamine neurons.

The psychostimulants cocaine and methamphetamine produce their euphoric effects through an interaction with the mesolimbic dopamine system. Methamphetamine, unlike cocaine, has been shown to be neurotoxic to both dopaminergic and serotonergic systems. We have previously determined that a 6 day exposure to methamphetamine causes neuronal damage to tyrosine hydroxylase-immunopositive cells in our tissue culture model of the mesencephalon. Over the same exposure period, cocaine neither impaired neuronal function nor altered dopamine cell survival. To test whether a longer exposure period to cocaine would alter dopamine function, we added cocaine (100 microM) to the cultures once daily for either 8 or 11 days and examined changes in dopamine uptake, cell survival and morphology 24 h after the last administration. Cocaine did not produce any signs of neurotoxicity in the mesencephalic cultures.

Stimulant-induced alterations in dopaminergic and serotonergic function in fetal raphe neurons.

Methamphetamine and its structural analogues have been demonstrated to be neurotoxic to CNS dopamine (DA) and serotonin (5-HT) neurons both in vivo and in vitro. Our laboratory has been actively characterizing mesencephalic cultures and the effects of methamphetamine exposure on neurochemical and immunochemical indices. The purpose of the present studies was to extend our findings with mesencephalic cultures and compare them with methamphetamine-induced alterations in fetal raphe cultures that contain both DA and 5-HT cells. Methamphetamine (10 and 100 microM) was added to the cultures 24 h after plating and fresh daily thereafter. The effects of chronic methamphetamine exposure on [3H]-DA and [3H]-5-HT uptake were determined after 5 days of drug treatment. Additional cultures were immunochemically analyzed for the presence of DA- and 5-HT-containing cells and total neuronal density. Results indicate that repeated methamphetamine exposure decreased DA and 5-HT uptake. Furthermore, repeated exposure to the higher concentration of methamphetamine (100 microM) caused a significant reduction in the number of DA and 5-HT cells as well as reducing the total neuronal density. This would suggest that this higher concentration of methamphetamine results in generalized neurotoxicity. Exposure to 10 microM methamphetamine resulted in more specific effects on dopaminergic function. These findings indicate that repeated methamphetamine administration can induce similar alterations in both dopaminergic and serotonergic neurons in raphe cultures.

Cannabinoid receptors: G-protein-mediated signal transduction mechanisms.

The recent discovery and cloning of cannabinoid receptors has provided a major breakthrough in the understanding of the biochemical mechanisms of action of delta 9-tetrahydrocannibinol (delta 9-THC). Cannabinoid receptors are coupled to G-proteins and inhibit adenylyl cyclase in a variety of systems. In the brain, cannabinoid-inhibited adenylyl cyclase and the receptors are particularly prevalent in the cerebellum, where they are localized to cerebellar granule cells (Fig. 1). In these cells, cannabinoid receptors are co-localized with other Gi/o-linked receptors such as gamma-aminobutyric acid (GABAB) receptors, where they share common effector systems (adenylyl cyclase catalytic units) but not common G-proteins. This sharing of effectors leads to the phenomenon of receptor convergence, in which agonists of different receptor types can produce the same biological response in certain cells. In cultured hippocampal neurons, cannabinoids also act through G-proteins to increase potassium conductance. In these cells, the predominant electrophysiological response at relatively low (microM) concentrations of cannabinoids is mediated through a voltage-sensitive potassium A current (IA) (Fig. 1). The action of cannabinoid receptors in this system is to shift the voltage sensitivity of IA channels to higher voltage ranges, thus increasing K+ conductance at lower membrane potentials and decreasing the probability of multiple action potentials. When combined with data from other groups showing a cannabinoid receptor-mediated decrease in calcium conductance, along with the unique localization of cannabinoid receptors in the brain, it is clear that these receptor-effector combinations are well situated to mediate many of the well-known neurobiological effects of delta 9-THC.

Cannabinoids modulate potassium current in cultured hippocampal neurons.

Characterization of the newly discovered G-protein-coupled cannabinoid receptor in brain requires determination of its functional significance. The effects are reported of several potent cannabinoid analogs (CP 55,244, CP 55,940, levonantradol and WIN 55,212-2) on cultured neurons from hippocampus, a brain region that exhibits high cannabinoid receptor density. The electrophysiological effects of cannabinoids were determined by whole-cell patch clamp recordings of voltage-dependent potassium currents. The voltage dependence of the rapidly inactivating potassium A current (IA), characteristic of hippocampal neurons, was significantly altered in a concentration-dependent manner by cannabinoid analogs. Decreased inactivation, which led to an increased activation of IA near resting levels in these cells, was observed after brief local extracellular applications of cannabinoids. These actions were blocked by pertussis toxin. Cellular dialysis of GTP-gamma-S mimicked the actions of cannabinoids on IA while blocking further effects due to added cannabinoids. The rank order of potency of the cannabinoid analogs was similar to that observed with respect to binding at cannabinoid receptors in brain membranes. The concentration-related effectiveness of cannabinoid analogs in modulating IA was similar to their potency in stimulating low Km GTPase in cell membranes isolated from the cannabinoid receptor-rich dentate gyrus. These data support the conclusion that cannabinoid effects on IA are mediated through G-protein-coupled receptors. This cannabinoid-induced shift in the voltage dependence of IA could serve to counteract fast, transient, depolarizing events such as action potentials and synaptic currents in hippocampal neurons.

An immunochemical and biochemical study of catecholaminergic activity in dissociated hypothalamic cultures.

Dopamine (DA)-containing cells of the medial basal hypothalamus (MBH) were dissociated and maintained in culture for up to 9 days. Cultures were evaluated both biochemically and immunochemically for DA activity. DA biosynthesis was determined using incorporation of [3H]tyrosine and was analyzed by HPLC with electrochemical detection. Immunochemical studies were performed to identify tyrosine hydroxylase (TH)-positive and neuron-specific enolase (NSE)-positive cells. Morphometric analyses determined the cell size, density, process length and the percent of neurons which were catecholaminergic. TH-positive neurons ranged from 6 to 8% of the total neuronal population when examined over days 3-9 of culture and the length of TH-positive neurites was significantly greater than that of NSE-positive cells. There was incorporation of [3H]tyrosine into DA as evidenced by the presence of [3H]DA in both the media and tissue and the inhibition of synthesis with alpha-methyl-p-tyrosine. There was a greater amount of labeled DA in the media than in the tissue at every time point examined. On the other hand, biosynthesis of DA in fresh brain tissue revealed approximately equal levels of DA in the media and tissue. These studies indicate that DA continues to be synthesized in dissociated cultures of MBH as evidenced by both the biochemical and immunochemical analyses and that there appears to be some alteration in the ability of these neurons to store the newly synthesized amine.