Cannabinoid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr(144) and Thr(370) were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.     

Regional muscle oxygenation differences in vastus lateralis during different modes of incremental exercise

Background

Near infrared spectroscopy (NIRS) is used to assess muscle oxygenation (MO) within skeletal muscle at rest and during aerobic exercise. Previous investigations have used a single probe placement to measure MO during various forms of exercise. However, regional MO differences have been shown to exist within the same muscle which suggests that different areas of the same muscle may have divergent MO. Thus, the aim of this study was to examine whether regional differences in MO exist within the same muscle during different types of incremental (rest, 25, 50, 75, 100 % of maximum) exercise (1 leg knee extension (KE), 2 leg KE, or cycling).

Methods

Nineteen healthy active males (Mean ± SD: Age 27 ± 4 yrs; VO_2max: 55 ± 11 mL/kg/min) performed incremental exercise to fatigue using each mode of exercise. NIRS probes were placed on the distal and proximal portion of right leg vastus lateralis (VL). Results were analyzed with a 3-way mixed model ANOVA (probe × intensity × mode).

Results

Differences in MO exist within the VL for each mode of exercise, however these differences were not consistent for each level of intensity. Comparison of MO revealed that the distal region of VL was significantly lower throughout KE exercise (1 leg KE proximal MO – distal MO = 9.9 %; 2 leg KE proximal MO – distal MO = 13 %). In contrast, the difference in MO between proximal and distal regions of VL was smaller in cycling and was not significantly different at heavy workloads (75 and 100 % of maximum).

Conclusion

MO is different within the same muscle and the pattern of the difference will change depending on the mode and intensity of exercise. Future investigations should limit conclusions on MO to the area under assessment as well as the type and intensity of exercise employed.

     

Mycobacterium tuberculosis ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability in vitro and During Infection

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy.     

锌对铅染毒新生大鼠成骨细胞增殖分化的影响

目的:探讨铅锌联合染毒对乳鼠颅骨成骨细胞增殖分化的影响。方法:分离并培养原代成骨细胞,加入不同浓度铅、锌培养48h,检测其对成骨细胞增殖的作用;用碱性磷酸酶试剂盒检测ALP活力。结果:在染铅48h后,当铅浓度≥10μmol/L时,细胞增殖功能下降(P<0.05);加锌干预48h后,铅+锌组细胞增殖功能均高于各自单独染铅组,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组、铅(10)+锌(100)组与对照组间的差异具有统计学意义(P<0.05)。铅干预48h后,100μmol/L铅组的ALP活力显著下(P<0.05),给予锌干预的铅锌联合染毒组,各组ALP活力均有增加,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组ALP活力均高于对照组,而铅(100μmol/L)+锌(50μmol/L)组ALP活力低于对照组,差异均有统计学意义(P<0.05)。结论:铅对成骨细胞有毒性作用,影响其增殖和分化功能;50μmol/L锌在一定程度上可以拮抗铅对成骨细胞增殖和分化功能的损伤,且对ALP活力的作用更显著,为铅中毒骨病的防治提供一定的科学依据。     

Morphology and coexistence of congeneric ectoparasite species: reinforcement of reproductive isolation?

Assuming that differences or similarities in morphology among congeneric parasite species living in the same habitat are not a random pattern, several hypotheses explaining morphological differences were tested: (i) reproductive isolation, (ii) niche restriction resulting from competition, and (iii) niche specialization. Congeneric monogenean (platyhelminth) ectoparasites parasitizing the gills of one host species were used as an ecological model. Morphometric distances of the attachment organ and morphometric distances of the copulatory organ between species pairs were calculated, Levin's niche size and Renkonen niche overlap indices were applied. Our results support the prediction that the function of niche segregation is to achieve reproductive isolation of related species in order to prevent hybridization (reinforcement of reproductive barriers). Parasite species living in the same niche differ greatly in the size of copulatory organ. Moreover, species coexistence is facilitated by an increase in morphometric distances of copulatory organ and niche centre distances. Our results also show that species living in overlapping niches have similar attachment organs, which supports the prediction that morphologically similar species have the same ecological requirements within one host and suggests small effects of interspecific competition for the evolution of morphological diversity of attachment organs. Specialist adaptations also seem to facilitate species coexistence and affect the niche distribution within host species. Parasite species that can colonize more than one host species, i.e. generalists, occupy more distant niches within host species than strictly host-specific parasites. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 76, 125–135.     

Effect of mediodorsal thalamic nucleus on the respiratory neurons from medulla oblongata and the rat respiration during hypoxia]

In the normal as well as in the oxygen deficiency conditions the research has been conducted to study the influence of associative mediodorsal (MD) nucleus of thalamus on impulsive activity of respiratory neurons of medulla oblongata of respiration. In conditions of normal atmospheric pressure, before the uplift of the animals, the electrical stimulation of MD of nucleus of thalamus has had mainly inhibiting influence. In the initial phase, on 4-5 thousand meter altitude, activation of frequent discharge of neurons occurred, the respiration has become frequent as well. In this situation the inhibiting influence of stimulation of MD nucleus of thalamus was more accentuated than in conditions of normoxia. In the second phase, 7.5-8 thousand meters, the opposite occurred, i.e. reduction of respiratory center activity of medulla oblongata and thalamus. In this difficult conditions of hypoxia, a reduction of impulsive activity of neurons has been observed; the respiration was becoming slower and surface. Meanwhile, the inhibiting influence of thalamus was not significant.     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.