Intraganglionic Signaling as a Novel Nasal-Meningeal Pathway for TRPA1-Dependent Trigeminovascular Activation by Inhaled Environmental Irritants

Headache is the most common symptom associated with air pollution, but little is understood about the underlying mechanism. Nasal administration of environmental irritants activates the trigeminovascular system by a TRPA1-dependent process. This report addresses questions about the anatomical pathway involved and the function of TRP channels in this pathway. TRPV1 and TRPA1 are frequently co-localized and interact to modulate function in sensory neurons. We demonstrate here that resiniferatoxin ablation of TRPV1 expressing neurons significantly reduces meningeal blood flow responses to nasal administration of both TRPV1 and TRPA1 agonists. Accordingly resiniferatoxin also significantly reduces TRPV1 and CGRP immunostaining and TRPV1 and TRPA1 message levels in trigeminal ganglia. Sensory neurons of the trigeminal ganglia innervate the nasal epithelium and the meninges, but the mechanism and anatomical route by which nasal administration evokes meningeal vasodilatation is unclear. Double retrograde labeling from the nose and meninges reveals no co-localization of fluorescent label, however nasal and meningeal labeled cells are located in close proximity to each other within the trigeminal ganglion. Our data demonstrate that TRPV1 expressing neurons are important for TRPA1 responses in the nasal-meningeal pathway. Our data also suggest that the nasal-meningeal pathway is not primarily by axon reflex, but may instead result from intraganglionic transmission.     

The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy

Replication-competent HIV-1 can be isolated from infected patients despite prolonged plasma virus suppression by anti-retroviral treatment. Recent studies have identified resting, memory CD4+ T lymphocytes as a long-lived latent reservoir of HIV-1 (refs. 4,5). Cross-sectional analyses indicate that the reservoir is rather small, between 103 and 107 cells per patient. In individuals whose plasma viremia levels are well suppressed by anti-retroviral therapy, peripheral blood mononuclear cells containing replication-competent HIV-1 were found to decay with a mean half-life of approximately 6 months, close to the decay characteristics of memory lymphocytes in humans and monkeys. In contrast, little decay was found in a less-selective patient population. We undertook this study to address this apparent discrepancy. Using a quantitative micro-culture assay, we demonstrate here that the latent reservoir decays with a mean half-life of 6.3 months in patients who consistently maintain plasma HIV-1 RNA levels of fewer than 50 copies/ml. Slower decay rates occur in individuals who experience intermittent episodes of plasma viremia. Our findings indicate that the persistence of the latent reservoir of HIV-1 despite prolonged treatment is due not only to its slow intrinsic decay characteristics but also to the inability of current drug regimens to completely block HIV-1 replication.     

Sensitive detection of specific repair endonucleases: radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31.0% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.     

Increased methionine synthetase activity in zinc-deficient rat liver

To study the effect of zinc deficiency on folate metabolism, three groups of male Sprague-Dawley rats (zinc deficient (ZD), restricted-fed (RF + Zn), and ad libitum-fed control (control] were given a semipurified 25% egg white protein diet. The ZD group received less than 10.3 nmol zinc/g of diet, while the RF + Zn and control groups were given 1620 nmol zinc/g of diet. After 6-7 weeks of feeding, severe zinc deficiency developed in ZD rats. Hepatic methionine synthetase activity was increased in the ZD group compared to both the RF + Zn and control groups, but hepatic 5,10-CH2-H4folate reductase activity was similar in all groups. This increased methionine synthetase activity found in zinc-deficient rats might induce secondary alterations in folate metabolism. These changes include significantly lowered plasma folate levels, decreased 5-CH3-H4folate in liver, and increased rates of histidine and formate oxidation. The latter two findings suggest that the available non-5-CH3-H4folate is increased in zinc deficiency.     

Analysis of whole blood manganese by flameless atomic absorption spectrophotometry and its use as an indicator of manganese status in animals

To investigate the use of whole blood manganese (Mn) as an indicator of total body Mn, we measured Mn in whole blood and liver of rats fed purified diets containing adequate (45 micrograms Mn/g diet) or deficient (1 microgram Mn/g diet) Mn. The mean hepatic Mn concentration was significantly lower (P less than 0.001) in the Mn-deficient group compared to the control group, 0.36, microgram Mn/g and 1.73 micrograms Mn/g, respectively. Furthermore, whole blood Mn was significantly reduced (P less than 0.001) in the deficient group when compared to the control group, 4.0 ng Mn/ml and 8.6 ng Mn/ml, respectively. Hepatic Mn linearly regressed against whole blood Mn yielded a statistically significant (P less than 0.001) correlation coefficient of 0.775. These data suggest that whole blood Mn is a valid indicator of body Mn status and thus may be useful, in addition to the measurements of serum copper and zinc, for the diagnosis and prognosis of diseases in which the metabolism of trace elements is affected. In addition, this paper describes and delineates operational parameters for the measurement of whole blood Mn using the IL 551 atomic absorption spectrophotometer and the IL 555 B flameless atomizer.     

Diel variability of mercury phase and species distributions in the Florida Everglades

Preliminary studies of mercury (Hg) cycling in the Everglades revealed that dissolved gaseous mercury (DGM), total mercury (Hg_T), and reactive mercury (Hg_R) show reproducible, diel trends. Peak water-column DGM concentrations were observed on or about noon, with a 3 to 7 fold increase over night-time concentrations. Production of DGM appears to cease during dark periods, with nearly constant water column concentrations that were at or near saturation with respect to the overlying air. A simple mass balance shows that the flux of Hg to the atmosphere from diel DGM production and evasion represents about 10% of the annual input from atmospheric deposition. Production of DGM is likely the result of an indirect photolysis reaction that involves the production of reductive species and/or reduction by electron transfer. Diel variability in Hg_T and Hg_R appears to be controlled by two factors: inputs from rainfall and photolytic sorption/desorption processes. A possible mechanism involves photolysis of chromophores on the surface of a solid substrate (e.g., the periphyton mat) giving rise to destabilization of sorbed mercury and net desorption during daylight. At night, the sorption reactions predominate and the water-column Hg_T decreases. Methylmercury (MeHg) also showed diel trends in concentration but were not clearly linked to the solar cycle or rainfall at the study site.     

Lectin staining of sheep microglia

The B₄-isolectin from Griffonia simplicifolia is known to stain microglial cells in a variety of species. The present report describes a lectin staining method that has been modified to facilitate staining of resting microglia, as well as perivascular cells, in vibratome sections of normal sheep brain. This modified method employs tissue fixed in formaldehyde or paraformaldehyde and requires incubating sections with Triton X-100 prior to staining.