Cutaneous Magnetic Stimulation Reduces Rat Chronic Pain via Activation of the Supra-Spinal Descending Pathway

Recent studies have demonstrated that magnetic stimulation (MS) can induce cellular responses such as Ca²⁺ influx into the cultured neurons and glia, leading to increased intracellular phosphorylation. We have demonstrated previously that MS reduces rat neuropathic pain associated with the prevention of neuronal degeneration. Thus, we aimed to elucidate the actions of MS in relation to modulation of spinal neuron–glia and the descending inhibitory system in chronic pain. The male SD rats intrathecally implanted with catheters were subjected to sciatic nerve ligation (CCI). MS is a low power apparatus characterized by two different frequencies, 2 KHz and 83 MHz. Rats were given MS to the skin (injured sciatic nerve) for 10 min from the seventh day after CCI. The paw withdrawal latency (PWL) evoked by thermal stimuli was measured for 14 days after CCI. Immunohistochemistry for Iba-1 or GFAP was performed after 4% paraformaldehyde fixation (microscopic analysis). We employed microdialysis for measuring CSF 5-HIAA as a reflection of 5-HT release by MS stimulation. Following CCI, rats showed a decrease in PWL after CCI, and the decrease continued until the 14th day. With MS treatment, the decrease in PWL was reduced during the 10–14 day after CCI. Injection of JNK-1 inhibitors on the 14th day antagonized the analgesic effect of MS. MS also eliminated the CCI-induced decrease in GFAP immunoreactivity. Moreover, MS evoked spinal 5-HT release reflected by increase in spinal 5-HIAA level. Thus, we demonstrate that a novel magnetic stimulator used cutaneously can ameliorate chronic pain by not only preventing abnormal spinal neuron–glia interaction, but also through the activation of the supra-spinal descending inhibitory system.     

Fragmented CagA protein is highly immunoreactive in Japanese patients

Background: High‐molecular‐weight cell‐associated proteins (HM‐CAP) assay is the most popular serological immunoassay worldwide and has been developed from US isolates as the antigens. The accuracy is reduced when the sera are from adults and children in East Asia including Japan. To overcome the reduced accuracy, an enzyme immunoassay using Japanese strain–derived HM‐CAP (JHM‐CAP) was developed, in which the antigens were prepared by exactly the same procedure as HM‐CAP. The performance of JHM‐CAP was better than that of HM‐CAP in Japanese adults as well as in children. The higher sensitivity was because of the presence of 100‐kDa protein that was absent in the preparation of HM‐CAP antigen. Materials and Methods: Immunoblot analysis and peptide mass fingerprinting methods were used to identify the distinctive 100‐kDa protein present in JHM‐CAP antigens. The peptide sequence and identification were analyzed by Mascot Search on the database of Helicobacter pylori. The identified protein was confirmed by immunoblot with a specific antibody and inhibition assay by the sera. Results: The distinctive 100‐kDa protein was a fragment of CagA derived from Japanese clinical isolates, and the sera of Japanese patients had strongly reacted to the protein, probably to the exposed epitope on the fragmented CagA. The fragmentation of CagA had occurred in the process of antigen preparation in Japanese isolates, not in US isolates even under the same preparation. Conclusion: The distinctive 100‐kDa protein was a fragment of CagA protein of H. pylori derived from Japanese clinical isolates, and Japanese patients including children are likely to react strongly to the exposed epitopes on fragmented CagA.     

The occurrence of HR1b in the venom of the snake Okinawa habu (Protobothrops flavoviridis)

This study describes the first isolation of hemorrhagic metalloproteinase HR1b from the venom of Okinawa habu, and its cDNA cloning. The substrate specificity of isolated HR1b definitely differed from that of HR1a, further supporting the presence of a metalloproteinase distinguishable from HR1a in the venom of Okinawa habu. The deduced amino acid sequence of HR1b showed 99.67% identity with HR1b of Amami habu, with only two amino acid residue replacements.     

Metabolic fluxes in the liver of rats bearing the Walker-256 tumour: influence of the circulating levels of substrates and fatty acids

Studies on fatty acid and amino acid metabolism in the liver of Walker-256 tumour-bearing rats have revealed several changes. Comparisons, however, have been based on experiments performed with non-physiological, frequently unrealistic, substrate concentrations. The aim of the present work was to examine the influence of physiological substrate concentrations on gluconeogenesis, ketogenesis and related parameters. Isolated livers were perfused and substrates were infused at concentrations that were reported to occur in healthy and tumour-bearing rats. Ketogenesis and the mitochondrial NADH/NAD+ ratio were smaller in the tumour-bearing condition at low (0.2 mM) and high (0.8 mM) oleate concentrations. In the absence of oleate, gluconeogenesis from alanine (0.7 mM) and gluconeogenesis plus the associated changes in oxygen uptake due to lactate/pyruvate (2/0.2 and 6/0.3 mM) were smaller in livers of tumour-bearing rats. However, the response of gluconeogenesis from lactate/pyruvate in livers of tumour-bearing rats to 0.8 mM oleate was more pronounced so that a trend towards normalization was apparent at high substrate and oleate concentrations. Gluconeogenesis from 0.7 mM alanine was not significantly changed by oleate in the tumour-bearing state; in the control condition, stimulation occurred at 0.2 mM oleate and inhibition at 0.8 mM oleate. This diminution almost equalized the hepatic alanine-dependent gluconeogenesis of both control and tumour-bearing rats. Ureogenesis was smaller in the tumour-bearing state and was not affected by oleate. It was concluded that the high concentrations of fatty acids and lactate/pyruvate, which predominate in rats bearing the Walker-256 tumour, could be effective in normalizing the gluconeogenic response of livers from tumour-bearing rats.     

5-Lipoxygenase-derived lipid mediators are not required for the development of NSAID-induced inflammatory bowel disease in IL-10-/- mice

Leukotrienes are potent lipid mediators derived from the metabolism of arachidonic acid by the enzyme 5-lipoxygenase (5-LO). Elevated levels of the proinflammatory leukotriene LTB(4) have been found in preclinical models of inflammatory bowel disease (IBD) as well as in colon tissue from individuals with IBD. We therefore determined the extent to which absence of 5-LO-derived lipid mediators would alter the colitis in IL-10(-/-) mice, a model of human IBD. IL-10(-/-)/5-LO(-/-) mice were generated and were healthy. Absence of 5-LO did not alter the development of spontaneous colitis in IL-10-deficient mice. We then evaluated the extent to which absence of 5-LO would alter the development of NSAID-induced colitis in IL-10(-/-) mice. Absence of 5-LO did not delay the onset or alter the severity of inflammation in NSAID-treated IL-10(-/-) mice. At an early time point, 3 days after NSAID treatment was initiated, a qualitative increase in the number of dendritic cells and CD4(+) T cells was noted in the colons of IL-10(-/-)/5-LO(-/-); however, this difference was no longer present after 14 days of NSAID treatment. Absence of 5-LO did not alter the degree of neutrophil infiltration into the in this model. Absence of 5-LO does not alter the development of IFN-gamma producing Th1-type CD4(+) T cells or IL-17 producing CD4(+) T cells. Absence of 5-LO-derived mediators did not alter the expression of the adhesion molecules ICAM-1 and P-selectin. Development of colitis in IL-10(-/-) mice was associated with increased levels of the 5-LO-derived anti-inflammatory lipoxin LXA(4). These studies demonstrate that 5-LO-derived leukotrienes are not required for the development or maintenance of spontaneous or NSAID-induced colonic inflammation in IL-10(-/-) mice.     

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).     

Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2

Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease.     

Endoplasmic reticulum quality control regulates the fate of transthyretin variants in the cell

The secretion of transthyretin (TTR) variants contributes to the pathogenesis of amyloidosis because they form aggregates in the extracellular environment. However, the mechanism of how TTR variants pass the quality control system in the endoplasmic reticulum (ER) has not yet been elucidated. We investigated here the mechanism of how TTR passes ER monitoring. Monomeric mutation introduced in TTRs (M-TTRs) resulted in the ER retention of amyloidogenic M-TTRs but not non-amyloidogenic M-TTRs. Retention of amyloidogenic M-TTRs induced the unfolded protein response and upregulated the expression of ER chaperones BiP and glucose-regulated protein (GRP) 94. Additionally, we showed that the ER-retained amyloidogenic M-TTRs are subject to ER-associated degradation. On the other hand, the amyloidogenic TTR variants and non-amyloidogenic M-TTRs were secreted normally. These findings suggest that unlike for wild-type TTR, the ER quality control system may differentially regulate the fate of the TTR variants and their monomeric counterparts.