Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nociceptive neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Activation of TRPA1 by environmental irritants such as mustard oil, allicin and acrolein causes acute pain. However, the endogenous ligands that directly activate TRPA1 remain elusive in inflammation. Here, we show that a variety of inflammatory mediators (15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), nitric oxide (NO), hydrogen peroxide (H(2)O(2)), and proton (H(+))) activate human TRPA1 heterologously expressed in HEK cells. These inflammatory mediators induced robust Ca(2+) influx in a subset of mouse DRG neurons. The TRP channel blocker ruthenium red almost completely inhibited neuronal responses by 15d-PGJ(2) and NO, but partially suppressed responses to H(2)O(2) and H(+). Functional characterization of site-directed cysteine mutants of TRPA1 in combination with labeling experiments using biotinylated 15d-PGJ(2) demonstrated that modifications of cytoplasmic N-terminal cysteines (Cys421 and Cys621) were responsible for the activation of TRPA1 by 15d-PGJ(2). In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H(2)O(2), the extent of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ(2). Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H(+) as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.     

Homing of mouse spermatogonial stem cells to germline niche depends on beta1-integrin

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.     

Mutation in keratin 18 induces mitochondrial fragmentation in liver-derived epithelial cells

Microtubules (MTs) and microfilaments (MFs) are known to modulate mitochondrial morphology, distribution and function. However, little is known evidence about the role of intermediate filaments (IFs) in modulating mitochondria except desmin. To investigate whether or not the IFs regulate mitochondrial morphology, distribution, and function, we manipulated the IFs of cultured epithelial cells to express a mutant keratin 18 (K18). In contrast to the filamentous expression of wild K18, mutant K18 induced aggregation of K8/18, showing no fine IF network in the cells. In mutant K18-transfected cells, the mitochondria were fragmented into small spheroids, although they were observed as mitochondrial fibers in un-transfected or wild K18-transfected cells. Fluorescence recovery after photobleaching of fluorescence-labeled mitochondria was markedly less in the mutant K18-transfected cells, although a significant recovery was confirmed in wild K18-transfected cells. These findings suggest that the IFs are important for the maintenance of normal mitochondrial structures.     

A ten-year summary of reproductive parameters for ring-tailed lemurs at berenty, madagascar

From 1989 to 1998, 204 live births were recorded for ring-tailed lemurs (Lemur catta) at Berenty, Madagascar. Excluding unknown birth dates, the peak month of birth was September, with 82.0% (146/178) occurring during this period. The offspring sex ratio (1∶1.19) was not significantly different from 1∶1, and there was no association with the mother's age. The first births occurred at the ages of 2 to 4 yr. The annual birth rate was very low at the age of 2 yr (11.1%), but increased thereafter: to 50.0% at the age of 3 yr, and to 75–85% at the age of 4 or more years. Multiple births were very rare, since only three sets of twins and one set of triplets were recorded. As for the interbirth interval, a one-year interval was the most common (92.2%). Infant mortality within the first year was 37.7% (77/204). Neonatal mortality within the first month accounted for 31.2% of all infant dealths.     

Structure and mechanical properties of wet-spun fibers made from natural cellulose nanofibers

Cellulose nanofibers were prepared by TEMPO-mediated oxidation of wood pulp and tunicate cellulose. The cellulose nanofiber suspension in water was spun into an acetone coagulation bath. The spinning rate was varied from 0.1 to 100 m/min to align the nanofibers to the spun fibers. The fibers spun from the wood nanofibers had a hollow structure at spinning rates of >10 m/min, whereas the fibers spun from tunicate nanofibers were porous. Wide-angle X-ray diffraction analysis revealed that the wood and tunicate nanofibers were aligned to the fiber direction of the spun fibers at higher spinning rates. The wood spun fibers at 100 m/min had a Young's modulus of 23.6 GPa, tensile strength of 321 MPa, and elongation at break of 2.2%. The Young's modulus of the wood spun fibers increased with an increase in the spinning rate because of the nanofiber orientation effect.     

Effects of internal diffusion on the lineweaver-Burk plots for immobilized enzymes

The effect of internal diffusion on the slope and the intercept of the LineweaverBurk plots for the immobilized enzyme was considered theoretically and it was found that the slope and the intercept are influenced not only by the dimensionless term M but also by the range of the dimensionless bulk substrate concentration ζ_b. The dependencies of the slope and the intercept on M and on the rate of ζ_b are shown graphically. Accurate estimations of M and the maximum velocity of the immobilized enzyme give the true, not apparent, Michaelis constant. It is shown that the linear correlations in the Lineweaver-Burk plots do not always coincide with the correlations for the estimation of M and the maximum velocity. It also is shown that large values of M may induce a serious error in the estimation of M with large values of ζ_b and in an estimation of the maximum velocity with small values of ζ_b.     

Glucose tolerance test in the Ainu

In an attempt to find out specific characteristics in carbohydrate metabolism in the Ainu, if any, glucose tolerance test was carried out. Between the Ainu and non-Ainu Japanese no differences were observed in the blood glucose, immunoreactive insulin and human growth hormone levels in the plasma after glucose load. Fasting levels of plasma FFA were significantly lower in the Ainu than in the Japanese. Changes in the FFA after glucose load were less in the Ainu, while the Japanese showed a marked rebound of the plasma FFA.

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Zusammenfassung Der Glukosetoleranztest wurde bei 10 Ainu und 20 Japanern (Studenten) durchgeführt. Signifikante Unterschiede im Blutzucker, immunreaktiven Insulin und humanen Wachstumshormon im Plasma wurden nach der Glukosegabe nicht gefunden. Die Nüchternwerte der freien Fettsäuren waren in den Ainu signifikant tiefer als bei den Japanern. Die Aenderungen der freien Fettsäuren nach Glukose waren bei den Ainu geringer, während die Japaner einen ausgeprägten "rebound effect" aufwiesen.

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Resume On a appliqué un test de tolérance à la glucose à 10 Aïnous et à 20 Japonais d'autres races (tous étudiants). On n'a pas décelé de différence significative après administration de glucose ni dans la teneur en sucre du sang, ni dans le taux d'insuline immunoréactive, ni dans les hormones humaines de croissance contenues dans le plasma. Les coefficients d'acides gras libres (FFA), après un jeûne ont été plus bas chez les Aïnous que chez les Japonais et cela de façon significative. La modification des acides gras libres après administration de glucose a été moins marquée chez les Aïnous alors que les Japonais présentaient un net "effet de rebondissement".

     

Plasma free fatty acid composition in Ainu and Japanese men

The ratio of oleic acid in the plasma FFA fraction was higher in the Japanese than in the Ainu. When glucose was administered, the proportion of oleic acid was reduced, while that of stearic acid increased in both groups. In the Japanese, moreover, a decrease in linoleic acid and increases in palmitic and myristic acids were also observed. As the plasma FFA level rose after injection of norepinephrine, the proportion of oleic acid increased and that of palmitic, stearic, myristic and lauric acids decreased, and the composition approached the pattern of depot fat. It was inferred that the difference observed in the plasma FFA composition between the Ainu and Japanese was attributable to the difference in the plasma levels of FFA which was significantly lower in the Ainu.

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Zusammenfassung Das Verhältnis von Oelsäure in der freien Fettsäurenfraktion (FFA) im Plasma war bei Japanern (n=9) höher als bei Ainus (n=5). Nach Glukose war das Oelsäure-Verhältnis vermindert, während in beiden Gruppen Stearinsäure anstieg. Bei den Japanern ergaben sich ausserdem ein Abfall der Linolensäure und Austiege der Palmitin- und Myristinsäure. Wenn der Plasma FFA-Spiegel nach Noradrenalin-Injektion anstieg, stieg der Oelsäureanteil und fielen die Palmitin-, Myristin- und Laurinsäureanteile und die Zusammensetzung war wie die von Depotfett. Es wird angenommen, dass die Unterschiede in der Zusammensetzung der Plasma FFA bei Japanern und Ainus Folge der unterschiedlichen FFA-Spiegel waren, die bei den Ainu signifikant tiefer lagen.

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Resume La proportion d'acide oleique dans l'ensemble des acides gras libres (FFA) du plasma sanguin est plus marquée chez les Japonais (n=9) que chez les Aïnous (n=5). La proportion d'acide oleique a diminuê après l'administration de glucose, car, dans les deux groupes, la teneur en acide stéarique s'est renforcée. On constante en outre une diminution de l'acide linoleique et une augmentation des acides palmitique et myristique. Lorsque, après injection de noradrénaline, le taux de FFA du plasma se relève, on constate une augmentation des quantités relatives de l'acide oleique au détriment de la part prise par les acides palmitique, myristique et laurique et leur composition est semblable aux dépots adipeux. On peut ainsi admettre que les différences de composition du FFA plasmatique découlent du taux dissemblable du FFA entre Aïnous et Japonais. Ce taux est significativement inférieur chez les premiers.