Evaporative isotope enrichment as a constraint on reach water balance along a dryland river.

Deuterium and oxygen-18 enrichment in river water during its transit across dryland region is found to occur systematically along evaporation lines with slopes of close to 4 in (2)H-(18)O space, largely consistent with trends predicted by the Craig-Gordon model for an open-water dominated evaporating system. This, in combination with reach balance assessments and derived runoff ratios, strongly suggests that the enrichment signal and its variability in the Barwon-Darling river, Southeastern Australia is acquired during the process of evaporation from the river channel itself, as enhanced by the presence of abundant weirs, dams and other storages, rather than reflecting inherited enrichment signals from soil water evaporation in the watershed. Using a steady-state isotope mass balance analysis based on monthly (18)O and (2)H, we use the isotopic evolution of river water to re-construct a perspective of net exchange between the river and its contributing area along eight reaches of the river during a drought period from July 2002 to December 2003, including the duration of a minor flow event. The resulting scenario, which uses a combination of climatological averages and available real-time meteorological data, should be viewed as a preliminary test of the application rather than as a definitive inventory of reach water balance. As expected for a flood-driven dryland system, considerable temporal variability in exchange is predicted. While requiring additional real-time isotopic data for operational use, the method demonstrates potential as a non-invasive tool for detecting and quantifying water diversions, one that can be easily incorporated within existing water quality monitoring activities.

Bidirectional transcription regulation of glial fibrillary acidic protein by estradiol in vivo and in vitro.

Glial fibrillary acidic protein (GFAP) expression shows cyclic variation in the rat hypothalamus and hippocampus during the normal estrous cycle. To elucidate the role of transcription in the regulation of GFAP, we examined levels of GFAP intron 1 by in situ hybridization in the hypothalamus and hippocampus of normal, cycling rats. On the afternoon of proestrus, when plasma estradiol levels are highest, GFAP transcription and messenger RNA were both increased in the arcuate nucleus of the hypothalamus and decreased in the outer molecular layer of the dentate gyrus. In the hilus of the hippocampus, neither GFAP transcription nor messenger RNA changed during the estrous cycle. In vitro, astrocytes showed bidirectional responses, such that estradiol treatment increased GFAP transcription in monotypic astrocytic cultures but decreased GFAP transcription in astrocytes cocultured with neurons. The functionality of an estrogen response element in the 5'-upstream region of the GFAP promoter was established by site-directed mutagenesis and binding of human recombinant estrogen receptor in gel shift assays. We conclude that estrogen may act directly upon astrocytes by estrogen receptor binding, and that the direction of the transcriptional response is influenced by astrocyte-neuron interactions.

Increased transcription of the astrocyte gene GFAP during middle-age is attenuated by food restriction: implications for the role of oxidative stress.

Glial fibrillary acidic protein (GFAP), an intermediate filament of astrocytes, shows increased expression during aging. Because we found that chronic food restriction retards the increase of GFAP mRNA in aging rats and because food restriction decreases the load of oxidized proteins and lipids in association with increased life span, we investigated the regulation of GFAP during oxidative stress and aging. First, we showed that food restriction decreased the transcription of GFAP in aging rats. This result generalizes effects of food restriction on age changes of transcription; whether transcription decreases during aging as in hepatic genes, or increases during aging as in astrocytic GFAP, food restriction attenuates the age change. Moreover, food restriction decreased microglial activation during aging, which suggested the hypothesis that GFAP expression is sensitive to oxidative stress. Because GFAP transcription in cultured glia is increased by oxidative stress in response to hydrogen peroxide and cysteamine whether or not microglia were present, we conclude that responses of GFAP to oxidative stress in astrocytes do not depend on microglial activation. The results implicate oxidative stress in the increased expression of GFAP during aging, but also in responses to brain injury.

Localization of cells preferentially expressing GAD(67) with negligible GAD(65) transcripts in the rat hippocampus. A double in situ hybridization study.

Two major forms of glutamic acid decarboxylase (GAD) are present in the mammalian brain, a 65-kDa isoform (GAD(65)) and a 67-kDa isoform (GAD(67)), and it is usually assumed that all GABAergic neurons contain both. The two forms have not yet been colocalized to the same neurons, because the GAD(65) protein is found almost exclusively in axon terminals, while GAD(67) is found predominantly in the cell body. Using double in situ hybridization (DISH) with both radioactive [35S] and non-radioactive (digoxigenin, DIG) probes, the distributions of GAD(65) and GAD(67) mRNA have been simultaneously examined in the rat hippocampus. The results suggest that [35S] radioprobes are slightly more sensitive than DIG probes, and that the reversal of labels is necessary in DISH studies to determine whether a neuronal subtype which expresses only one isoform of GAD may be present. The data indicate that the majority of cells (90%) showing labeling were labeled for both GAD(65) and GAD(67) mRNA. In sectors CA1 and CA3 approximately 5-10% of the cells positive for GAD(67) showed little or no detectable GAD(65) mRNA. In the hilus, however, GAD(65) levels were higher, and all cells seem to express both GAD(65) and GAD(67) mRNA. Taken together, these results support the view that most GABAergic neurons in the hippocampus express both GAD(65) and GAD(67). However, it appears that some interneurons in the CA subfields differ from "classic" GABAergic interneurons by preferentially expressing the 67-kDa isoform of GAD under baseline conditions, with GAD(65) mRNA levels very low or absent.

Persistence of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. Evaluation of therapy by follow-up transbronchial lung biopsy.

The effectiveness of therapy with trimethoprim-sulfamethoxazole and/or pentamidine has not been fully evaluated in AIDS patients with Pneumocystis carinii pneumonia (PCP). Since recurrence of PCP is common, follow-up lung biopsy (15 transbronchial, one open) was performed as part of the clinical evaluation of 16 episodes of PCP. All patients had shown evidence of clinical improvement during treatment and had received a mean duration of therapy of 17.6 days. In six of 16 episodes (38 percent), however, a repeat biopsy remained positive for PCP an average of 25.2 days after initial diagnosis. Retrospective comparison of standard clinical parameters (fever response, arterial blood gas results, roentgenographic characteristics) could not adequately distinguish episodes with positive follow-up biopsies from those with negative biopsies. Persistence of PCP after conventional treatment may be an important factor in recurrences of this infection in AIDS patients.

Roentgenographic patterns of Pneumocystis carinii pneumonia in 104 patients with AIDS.

We reviewed the initial and follow-up chest roentgenograms (CXR) of 104 patients with the acquired immune deficiency syndrome (AIDS) and Pneumocystis carinii pneumonia (PCP) diagnosed between 1981 and 1985 in order to determine the relative frequencies of its various roentgenographic patterns. Although a diffuse bilateral interstitial infiltrate is most common, it was concluded that unusual and atypical roentgenographic manifestations of PCP occur in AIDS. These include localized infiltrate, cystic or honeycomb lesions, hilar enlargement and spontaneous pneumothorax.

Could dual G-protein coupling explain [D-Met2]FMRFamide's mixed action in vivo?

In vitro studies have demonstrated that FMRFamide-related peptide receptors can be coupled to different G-proteins, mediating opposite stimulatory and inhibitory effects. The present study tested whether this duality might extend to effects in vivo. Antinociception in mice of ICV [D-Met2]FMRFamide, which produced agonist [ED50 = 36.3 micrograms (61.6 nmol)] and antagonist [ID50 = 0.72 microgram (1.22 nmol)] actions, was attenuated by 24-h pretreatment with ICV pertussis toxin (ID50 = 0.55 microgram) or cholera toxin (ID50 = 0.09 microgram), suggesting that [D-Met2]FMRFamide in vivo effects might also be explained by dual Gi/Gs, coupling.

Mu receptor and Gi2alpha antisense attenuate [D-Met2]-FMRFamide antinociception in mice.

FMRFamide (Phe-Met-Arg-Phe-NH2) and several analogs produce centrally-mediated, naloxone-reversible antinociception, but have minimal affinity for opioid receptor (sub)types. In the present study, the antinociception in mice (55 degrees C tail-flick test) produced by supraspinal (intracerebroventricular; i.c.v.) administration of [D-Met2]-FMRFamide (a stable analog of FMRFamide) was attenuated by pretreatment with i.c.v. oligodeoxyribonucleotide antisense to the opioid mu receptor or by antisense to the Gi2alpha G-protein subunit. These data suggest that [D-Met2]-FMRFamide produces its antinociception via an opioid interneuron.

Unexpected and pronounced antinociceptive synergy between spinal acetaminophen (paracetamol) and phentolamine.

Acetaminophen was administered to mice by spinal (intrathecal, i.t.) injection alone or with phentolamine (11.3 microg = 0.03 micromol). Acetaminophen produced dose-related antinociception in the abdominal irritant test with an ED(50) value of 137.2 microg (0.9 micromol) Phentolamine had no effect. For combined administration, the potency of acetaminophen was significantly increased (ED50=24.4 vs. 137.2 microg), indicative of multiplicative interaction and strong synergism. These results reveal the significant and surprising interaction of spinal cord adrenoceptors or ion channel subtypes with acetaminophen-induced antinociception.

Differential cholera-toxin sensitivity of supraspinal antinociception induced by the cannabinoid agonists delta9-THC, WIN 55,212-2 and anandamide in mice.

Intracerebroventricular (i.c.v.) administration to mice of delta9-tetrahydrocannabinol (delta9-THC), WIN 55,212-2 or the endogenous cannabinoid anandamide induced dose-related antinociception in the 55 degrees C warm-water tail-flick test. Pretreatment (24 h, i.c.v.) with pertussis toxin dose-dependently reduced the antinociceptive effect of delta9-THC (955 nmol), WIN 55,212-2 (30 nmol) and anandamide (135 nmol) (IC50 = 0.13, 5.5, and 0.32 nmol, respectively). In contrast, pretreatment (24 h, i.c.v.) with cholera toxin (0.1-3.0 mg) reduced the antinociception of WIN 55,212-2, had minimal effect on delta9-THC, and dose-dependently increased the antinociception of anandamide (ED50 = 0.50 nmol). These data suggest differences in the receptor-effector coupling of delta9-THC, WIN 55,212-2 and anandamide in supraspinal-induced antinociception in mice.

Transcription supports age-related increases of GFAP gene expression in the male rat brain.

During aging, rodent and human brains show progressive increases in the levels of glial fibrillary acidic protein (GFAP) mRNA and protein. The role of transcription was investigated by in situ hybridization, using an intron-containing cRNA probe as a measure of primary GFAP transcripts. We found parallel age-related increases in GFAP intron RNA in the hippocampus, internal capsule, and corpus callosum of 3 versus 24 month old male F344 rats. We conclude that increased transcription supports the age-related increase of GFAP mRNA and protein. GFAP is a unique example of a gene that shows increased expression during aging in contrast to the decreased transcription of certain genes reported in non-neural tissues.

Determination of the adsorption of tramadol hydrochloride by activated charcoal in vitro and in vivo.

Although tramadol is one of the most widely used centrally acting analgesics worldwide, no literature is available regarding adsorption of tramadol HCl powder or tablets (Ultram; 50 mg tramadol HCl per tablet) by activated charcoal (AC) for use as potential adjunct treatment of overdose. The present study incorporated a novel combination of in vitro and in vivo methods to investigate this question. Based on a binding curve of tramadol UV absorbance (UV(a); 225 nm) plotted against the amount of AC, the ratio of amount of tramadol completely adsorbed by AC was 0.05 mg/mg. Also based on UV(a), no tramadol was detected in filtrate of slurries in which up to 62 tablets of Ultram were mixed with 50 g AC; 4.6% of unbound tramadol was detected when 100 tablets of Ultram were mixed with AC. The ratio of amount of tramadol completely adsorbed by AC in this test was 0.10. In vivo, co-administration of 0.1 g/ml of AC produced a 13- to 14-fold rightward shift in tramadol's antinociceptive dose-response curve and a 1.6-fold rightward shift in tramadol's lethality dose-response curve.

Efficient designs for studying synergistic drug combinations.

Distinguishing between pharmacologically additive and synergistic drug combinations requires experimental designs and statistical analyses that often require appreciable numbers of animals and much experimenter time. The current study employed a design in which individual dose-effect data from each drug were translated into theoretically additive total dose combinations, in a fixed drug proportion, in order to produce a composite additive dose-effect relation that could be compared with that of an actual mixture having the same proportion. Results from this approach, using a combination of intrathecal doses of morphine and clonidine, were virtually identical to those using isobolographic analysis of the same data set. Both analyses showed significant synergism for this combination and, in each method, it was not necessary to constrain the drug regression lines to parallelism. In contrast to the isobole approach, the use of the composite additive dose-effect relation also allows observation of the interaction over a range of effects while reducing the size of the data sets needed.

Alpha-subunit G-protein antisense oligodeoxynucleotide effects on supraspinal (i.c.v.) alpha2-adrenoceptor antinociception in mice.

An in vivo antisense strategy was used to examine the involvement of G-protein subunits in supraspinal (intracerebroventricular; i.c.v.) alpha2-adrenoceptor-mediated antinociception. Mice that were injected with 33-mer antisense oligodeoxyribonucleotides (6 nmol) or vehicle were tested (tail-flick) with an agonist (clonidine, guanfacine or BH-T 920) administered i.c.v. 18 - 24 h later. Gi3alpha antisense treatment attenuated BH-T 920 and clonidine-induced antinociception. Gi2alpha antisense produced differential effects on the three agonists. Gi1alpha and G(s)alpha antisense treatment had no significant effect. Together with the previous demonstration that i.c.v. mu-opioid antinociception is mediated via Gi2alpha, the present results suggest that different receptors may mediate antinociception via different G-protein subunits and, hence, that specific subunits might offer novel targets for drug discovery.

The effects of proprandolol and histamine on the ventilation response to carbon dioxide inhalation in normal subjects.

A method was developed to measure the oxygen cost of ventilation during CO2 rebreathing. In 20 healthy normal subjects SGaw, MMEF, and FEV1 were measured prior to and following the infusion of propranolol. In five of the same subjects airway constriction was induced by inhalation of histamine. The use of both agents was followed by a significant decrease in the ventilation response to carbon dioxide inhalation. Even more significantly, the oxygen cost of the increase in ventilation measured during CO2 rebreathing rose significantly following either propranolol or histamine.

Comparison of the ventilation response to CO2 rebreathing in healthy smokers and nonsmokers and subjects with bronchitis.

A comparison of ventilatory tests and the response to carbon dioxide rebreathing was made in healthy smokers, nonsmokers, and in subjects with bronchitis. The response to carbon dioxide (CO2) rebreathing was the same in the healthy population but diminished in the bronchitic group. Effects of smoking on maximal expiratory flow rates did not correlate with the results of carbon dioxide rebreathing. A consistent pattern of relationship of ventilation response to carbon dioxide and age of the subject was apparent.