Prostaglandin E2 attenuates preoptic expression of GABAA receptors via EP3 receptors

Prostaglandin E(2) (PGE(2)) has been shown to produce fever by acting on EP3 receptors within the preoptic area of the brain. However, there is little information about the molecular events downstream of EP3 activation in preoptic neurons. As a first step toward this issue, we examined PGE(2)-induced gene expression changes at single-cell resolution in preoptic neurons expressing EP3. Brain sections of the preoptic area from PGE(2)- or saline-injected rats were stained with an anti-EP3 antibody, and the cell bodies of EP3-positive neurons were dissected and subjected to RNA amplification procedures. Microarray analysis of the amplified products demonstrated the possibility that gene expression of gamma-aminobutyric acid type A (GABA(A)) receptor subunits is decreased upon PGE(2) injection. Indeed, we found that most EP3-positive neurons in the mouse preoptic area are positive for the alpha2 or gamma2 GABA(A) receptor subunit. Moreover, PGE(2) decreased the preoptic gene expression of these GABA(A) subunits via an EP3-dependent and pertussis toxin-sensitive pathway. PGE(2) also attenuated the preoptic protein expression of the alpha2 subunit in wild-type but not in EP3-deficient mice. These results indicate that PGE(2)-EP3 signaling elicits G(i/o) activation in preoptic thermocenter neurons, and we propose the possibility that a rapid decrease in preoptic GABA(A) expression may be involved in PGE(2)-induced fever.     

Two conformational states of Ras GTPase exhibit differential GTP-binding kinetics

Previous ³¹P NMR studies revealed that small GTPases H-Ras and K-Ras in complex with GTP assume two interconverting conformational states, state 1 and state 2. While state 2 corresponds to an active conformation, little is known about the function of state 1, an inactive conformation incapable of effector binding. To address the biochemical properties of state 1, we measured the ³¹P NMR spectra of five Ras family small GTPases; H-Ras, M-Ras, Rap1A, Rap2A and RalA, and find that they exhibit distinctive state 2/state 1 populations with the ratios ranging from 0.072 for M-Ras to 16 for Rap2A. Further, we show that GTPases with higher populations of state 1 exhibit higher dissociation and association rate constants for GTP. These results imply that GTP loading to the nucleotide-free small GTPases preferentially yields state 1, which is subsequently converted to state 2, rendering the GTP-bound form functional.     

Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by alpha- and gamma-secretase

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer’s disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by α-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by γ-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by α- and γ-secretases, determining the expression level of AICD.     

The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China

The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 μl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.     

Parturition and recruitment of macrophages in cervix of mice lacking the prostaglandin F receptor

Parturition does not occur in transgenic mice lacking the prostaglandin F receptor (Ptgfr(-/-)) because luteolysis is forestalled and progesterone production persists. Ovariectomy of pregnant Ptgfr(-/-) mice leads to a decline in circulating progesterone and delivery of live pups. The objective of the present study was to test the hypothesis that immigration of macrophages and increased innervation of the cervix of Ptgfr(-/-) mice was associated with ripening and parturition. The cervix of pregnant Ptgfr(-/-) mice was studied on Days 15-21 after breeding; additional groups were ovariectomized on Day 19 of pregnancy, and the cervix obtained on Day 20 of pregnancy before birth or the next day at about 24 h after birth. On Days 18-19 of pregnancy, macrophage numbers and nerve fiber density increased more than 3-fold compared with findings in nonpregnant or Day 15 or 21 pregnant Ptgfr(-/-) mice. The magnitude and time course of these changes were comparable to those found in wild-type controls that delivered on Day 19 after breeding. Thus, the mechanism regulating macrophage immigration, innervation, and cervical remodeling in Ptgfr(-/-) mice with delayed parturition is similar to wild-type controls that deliver at term. By contrast, ovariectomy forestalled the decrease in cervical macrophages in Ptgfr(-/-) mice. By Day 21 after breeding, macrophage numbers more than double those after ovariectomy, relative to those found in pregnant Ptgfr(-/-) mice, whereas nerve fiber density was the same regardless of birth. Density of collagen structure in these mice directly matched macrophage traffic in the cervix. The findings indicate that the prostaglandin F(2alpha) receptor and progesterone withdrawal are a necessary part of the final common pathway for ripening of the cervix and the process of parturition.     

Points of control exerted along the macrophage-endothelial cell-polymorphonuclear neutrophil axis by PECAM-1 in the innate immune response of acute colonic inflammation

PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1(-/-) mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1(-/-) mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (MPhi) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although MPhi activation (NF-kappaB nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1(-/-) MPhi, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1(-/-) PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1(+/+) PMN, PECAM-1(-/-) PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.     

Statins modulate the levels of osteoprotegerin/receptor activator of NFkappaB ligand mRNA in mouse bone-cell cultures.

Statins stimulate bone formation partly by inducing osteoblast differentiation, although there is controversy about the effects of statins on bone mineral density and fracture risk. Several studies have revealed that statins suppress bone resorption. However, the mechanism by which statins inhibit bone resorption is still unclear. The present study was performed to clarify the effects of statins on osteoclast formation as well as the levels of osteoprotegerin (OPG) and receptor activator of NFkappaB ligand (RANKL) mRNA in mouse bone-cell cultures by semiquantitative RT-PCR. 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] significantly stimulated osteoclast formation and 10(-6) M statins (mevastatin and simvastatin) significantly antagonized osteoclast formation stimulated by 1,25(OH)2D3 in mouse bone-cell cultures, including both osteoblasts and osteoclasts. 10(-6) M mevastatin and simvastatin increased the level of OPG mRNA in mouse bone-cell cultures. On the other hand, 10(-6) M mevastatin and simvastatin inhibited the level of RANKL mRNA in these cultures. In conclusion, the present study demonstrates that statins inhibit osteoclast formation in mouse bone-cell cultures. Moreover, statins also increased and decreased the levels of OPG and RANKL mRNA expression in these cultures, respectively. The modulation of OPG/RANKL may be involved in the inhibition of osteoclast formation by statins.