Expression of the transient receptor potential channels TRPV1, TRPA1 and TRPM8 in mouse trigeminal primary afferent neurons innervating the dura

ABSTRACT: BACKGROUND: Migraine and other headache disorders affect a large percentage of the population and cause debilitating pain. Activation and sensitization of the trigeminal primary afferent neurons innervating the dura and cerebral vessels is a crucial step in the "headache circuit". Many dural afferent neurons respond to algesic and inflammatory agents. Given the clear role of the transient receptor potential (TRP) family of channels in both sensing chemical stimulants and mediating inflammatory pain, we investigated the expression of TRP channels in dural afferent neurons. METHODS: We used two fluorescent tracers to retrogradely label dural afferent neurons in adult mice and quantified the abundance of peptidergic and non-peptidergic neuron populations using calcitonin gene-related peptide immunoreactivity (CGRP-ir) and isolectin B4 (IB4) binding as markers, respectively. Using immunohistochemistry, we compared the expression of TRPV1 and TRPA1 channels in dural afferent neurons with the expression in total trigeminal ganglion (TG) neurons. To examine the distribution of TRPM8 channels, we labeled dural afferent neurons in mice expressing farnesylated enhanced green fluorescent protein (EGFPf) from a TRPM8 locus. We used nearest-neighbor measurement to predict the spatial association between dural afferent neurons and neurons expressing TRPA1 or TRPM8 channels in the TG.Results and conclusionsWe report that the size of dural afferent neurons is significantly larger than that of total TG neurons and facial skin afferents. Approximately 40% of dural afferent neurons exhibit IB4 binding. Surprisingly, the percentage of dural afferent neurons containing CGRP-ir is significantly lower than those of total TG neurons and facial skin afferents. Both TRPV1 and TRPA1 channels are expressed in dural afferent neurons. Furthermore, nearest-neighbor measurement indicates that TRPA1-expressing neurons are clustered around a subset of dural afferent neurons. Interestingly, TRPM8-expressing neurons are virtually absent in the dural afferent population, nor do these neurons cluster around dural afferent neurons. Taken together, our results suggest that TRPV1 and TRPA1 but not TRPM8 channels likely contribute to the excitation of dural afferent neurons and the subsequent activation of the headache circuit. These results provide an anatomical basis for understanding further the functional significance of TRP channels in headache pathophysiology.     

Family meal frequency and weight status among adolescents: cross-sectional and 5-year longitudinal associations

This study examined cross-sectional and 5-year longitudinal associations between the frequency of family meals and overweight status (>85th percentile for age and gender) in a large, diverse population of adolescents (n = 2,516). The population included two cohorts (midadolescence to young adulthood, n = 1,710, and early adolescence to midadolescence, n = 806). Logistic regression models tested cross-sectional and longitudinal (1999-2004) associations between family meal frequency and overweight status. Two sets of models are presented: (i) models adjusted only for baseline demographic characteristics and (ii) models also adjusted for physical activity, sedentary behaviors, and energy intake. Longitudinal models adjusted for baseline overweight status. Although significant inverse associations between family meal frequency and overweight status were observed for early adolescent females in all cross-sectional models (P < 0.001), longitudinal associations were not significant. Neither cross-sectional nor longitudinal associations were significant for males of either cohort and older females in any models. Young adolescent females who do not eat meals with their families may be at risk for overweight; however, the increased risk may not persist over a 5-year period. Eating family meals during high school may not protect against overweight during young adulthood. Although previous longitudinal research has suggested significant dietary and psychosocial benefits related to family meal frequency, the weight-related benefits of family meals may be more complex and deserving of further study, including an examination of the quality and quantity of food consumed at family meals.     

THE AXOSTYLE OF SACCINOBACULUS : I. Structure of the Organism and Its Microtubule Bundle

The axostyle of the flagellate Saccinobaculus is a motile ribbon composed of microtubules, cross-bridged to form interconnected rows. We find a centriole-related row of dark-staining tubules near the nucleus at the anterior end of the axostyle. Other tubule rows bind parallel to this primary row, acquire ordered relationships, and become the tubules of the axostyle proper. The number of tubule rows is constant in Saccinobaculus lata from the region near the nucleus to within a few micrometers of the posterior tip of the cell. In Saccinobaculus ambloaxostylus a few tubule rows are added to the axostyle posterior to the nucleus, giving this axostyle a leaf spring construction. The tubules of S. lata are held in rows by links with a 140 Å periodicity along the tubule axis; bridges between rows of tubules are also seen but are not apparently periodic. Each tubule in S. ambloaxostylus shows an axial periodicity of 150 Å due to pairs of arms, one of which is always part of the intrarow link. Interrow bridges in this species run either from tubule to tubule or from tubule to the free arm, but as in S. lata they do not display an obvious axial periodicity. An average unit cell is presented for the axostyle of each species, and the relation of the intertubule links to the microtubule substructure is discussed.     

Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of L-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 +/- 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100 degrees C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100 degrees C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.     

Monitoring cucumber beetles in sweetpotato and cucurbits with kairomone-baited traps

Seven kairomone formulations (Trécé, Inc., Salinas, CA) were evaluated for their effectiveness as attractants for luring three species of cucumber beetles into Pherocon CRW traps (Trécé, Inc.) in cucurbit and sweetpotato fields. The spotted cucumber beetle, Diabrotica undecimpunctata howardi Barber; the banded cucumber beetle, Diabrotica balteata LeConte; and the striped cucumber beetle, Acalymma vittatum (F.), were captured in this study. TRE8276 (TIC mixture: 500 mg of 1,2,4-trimethoxybenzene, 500 mg of indole, and 500 mg of trans-cinnamaldeyde) and TRE8336 (500 mg of 1,2,4-trimethoxybenzene, 500 mg of trans-cinnamaldeyde, 500 mg of 4-methoxyphenethanol) were the most effective lures for spotted and striped cucumber beetles. None of the kairomone lures was very effective for attracting banded cucumber beetles. Three population peaks of spotted cucumber beetles were observed in cucurbit and sweetpotato fields at the U.S. Vegetable Laboratory (Charleston, SC). The efficacy of TRE8276 declined rapidly after 2 wk in the field. An improved design of the Pherocon CRW trap, with a yellow bottom and more-tapered top section, was more effective for capturing cucumber beetles than the original trap design made entirely of clear plastic. Banded cucumber beetles were not captured in sweetpotato fields at inland locations in North Carolina or South Carolina.     

The shunt pathway of mevalonate metabolism in the isolated perfused rat kidney

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated kidneys from rats perfused with physiological concentrations of variously labeled [14C]- and [3H]mevalonates. The rate of operation of the shunt pathway was quantified by the production of either 14CO2 or 3H2O from the tracers. The measured rates of 14CO2 production from [14C] mevalonate were converted to rates of mitochondrial acetyl-CoA production by methods which take into account underestimations of metabolic rates derived from 14CO2 production. We have shown that the sex difference in renal shunting of mevalonate (Wiley, M. H., Howton, M. M., and Siperstein, M. D. (1979) J. Biol. Chem. 254, 837-842) occurs at physiological levels of substrate. The shunt pathway diverts up to 17% of the flux of mevalonate entering the cholesterol synthesis pathway in the kidney. It may, therefore, play a role in the long term regulation of cholesterol synthesis in this organ, as had been hypothesized by Edmond and Popják.     

Numbing the senses: role of TRPA1 in mechanical and cold sensation

In this issue of Neuron, Kwan et al. demonstrate that TRPA1 is critical for the transduction of noxious cold and mechanical stimuli, as well as in mediating the activation of nociceptors by endogenous and natural irritants. Differences between the present report and a previous study indicate that further study is needed to reach a consensus on the role of TRPA1 in the transduction of mechanical and noxious cold stimuli.     

Cultivar Variations of Australian‐Grown Danshen (Salvia miltiorrhiza): Bioactive Markers and Root Yields

Danshen (Salvia miltiorrhiza Bunge, Lamiaceae) is a commonly used and highly valued Chinese medicinal herb grown widely in China. In the present work, we studied cultivar variations of Australian‐grown Danshen in order to select optimal cultivars for local herbal production. Root yields of seven cultivars, V1–V7, were monitored in a one‐year field trial, and bioactive markers, including cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B, were quantitatively determined using a validated RP‐HPLC method. Significant variations were found in root yields, root production efficiencies, and contents of the bioactive marker compounds. Linear correlations were observed among the contents of three tanshinones but not among those of tanshinones and salvianolic acid B. Among the cultivars, V6 was the best cultivar for production of tanshinones, and V4 and V5 were best for production of salvianolic acid B. The findings indicate that it is possible to achieve optimal root yields, and high contents of tanshinones and salvianolic acid B by selecting specific Danshen cultivars.