Cytosolic acidification and intracellular zinc release in hippocampal neurons

In neurons exposed to glutamate, Ca2? influx triggers intracellular Zn2? release via an as yet unclear mechanism. As glutamate induces a Ca2?-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca2? concentration ([Ca2?](i)), intracellular pH (pH(i) ), and [Zn2?](i). Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn2?](i), [Ca2?](i) and pH(i) were monitored using FluoZin-3, Fura2-FF, and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pH(i) to 6.1 and induced intracellular Zn2? release in a Ca2?-dependent manner, as expected. The pH(i) drop reduced the affinity of FluoZin-3 and Fura-2-FF for Zn2?. The rate of Glu/Gly-induced [Zn2?](i) increase was not correlated with the rate of [Ca2?](i) increase. Instead, the extent of [Zn2?](i) elevations corresponded well to the rate of pH(i) drop. Namely, [Zn2?](i) increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca2?-dependent pH(i) drop (plasmalemmal Ca2? pump and mitochondria) counteracted the Glu/Gly-induced intracellular Zn2? release. Alkaline pH (8.5) suppressed Glu/Gly-induced intracellular Zn2? release whereas acidic pH (6.0) enhanced it. A pH(i) drop to 6.0 (without any Ca2? influx or glutamate receptor activation) led to intracellular Zn2? release; the released Zn2? (free Zn2? plus Zn2?) bound to Fura-2FF and FluoZin-3) reached 1 μM.     

Effects of different acute hypoxic regimens on tissue oxygen profiles and metabolic outcomes

Obstructive sleep apnea (OSA) causes intermittent hypoxia (IH) during sleep. Both obesity and OSA are associated with insulin resistance and systemic inflammation, which may be attributable to tissue hypoxia. We hypothesized that a pattern of hypoxic exposure determines both oxygen profiles in peripheral tissues and systemic metabolic outcomes, and that obesity has a modifying effect. Lean and obese C57BL6 mice were exposed to 12 h of intermittent hypoxia 60 times/h (IH60) [inspired O? fraction (Fi(O?)) 21-5%, 60/h], IH 12 times/h (Fi(O?) 5% for 15 s, 12/h), sustained hypoxia (SH; Fi(O?) 10%), or normoxia while fasting. Tissue oxygen partial pressure (Pti(O?)) in liver, skeletal muscle and epididymal fat, plasma leptin, adiponectin, insulin, blood glucose, and adipose tumor necrosis factor-α (TNF-α) were measured. In lean mice, IH60 caused oxygen swings in the liver, whereas fluctuations of Pti(O?) were attenuated in muscle and abolished in fat. In obese mice, baseline liver Pti(O?) was lower than in lean mice, whereas muscle and fat Pti(O?) did not differ. During IH, Pti(O?) was similar in obese and lean mice. All hypoxic regimens caused insulin resistance. In lean mice, hypoxia significantly increased leptin, especially during SH (44-fold); IH60, but not SH, induced a 2.5- to 3-fold increase in TNF-α secretion by fat. Obesity was associated with striking increases in leptin and TNF-α, which overwhelmed effects of hypoxia. In conclusion, IH60 led to oxygen fluctuations in liver and muscle and steady hypoxia in fat. IH and SH induced insulin resistance, but inflammation was increased only by IH60 in lean mice. Obesity caused severe inflammation, which was not augmented by acute hypoxic regimens.     

Effects of estrogen on endothelial prostanoid production and cyclooxygenase-2 and heme oxygenase-1 expression

We studied the effects of 17β-estradiol (E?) (10, 40 nM) on 2 vasoprotective pathways, i.e. cyclooxygenase-2 (COX-2)-dependent prostanoids and the antioxidant heme oxygenase-1 (HO-1), in human umbilical vein endothelial cells (HUVEC) exposed for 6h to steady laminar shear stress (LSS, 10 dyn/cm2), characteristic of atherosclerotic lesion-protected areas. COX-2 was induced by LSS versus static condition (SC). E? did not significantly affect COX-2 expression in HUVEC cultured in SC or exposed to LSS. Prostacyclin (PGI?) and prostaglandin (PG)E? were induced while PGF(2α) was reduced by LSS. E? caused no effect or a small reduction of prostanoid biosynthesis. In HUVEC cultured in SC or exposed to LSS, E? 10 nM caused a comparable HO-1 induction (35-45%) while E? 40 nM was 5-fold more potent in LSS-exposed HUVEC than in SC (290% and 58%, respectively). PGI? receptor antagonist RO3244794 did not affect HO-1 induction by E?. In conclusion, E? may restrain oxidant stress in the endothelium through HO-1 induction by a mechanism independent on PGI? signaling.     

Neuromodulating Attention and Mind-Wandering Processes with a Single Session Real Time EEG

Our minds are continuously alternating between external attention (EA) and mind wandering (MW). An appropriate balance between EA and MW is important for promoting efficient perceptual processing, executive functioning, decision-making, auto-biographical memory, and creativity. There is evidence that EA processes are associated with increased activity in high-frequency EEG bands (e.g., SMR), contrasting with the dominance of low-frequency bands during MW (e.g., Theta). The aim of the present study was to test the effects of two distinct single session real-time EEG (rtEEG) protocols (SMR up-training/Theta down-training—SMR?Theta?; Theta up-training/SMR down-training—Theta?SMR?) on EA and MW processes. Thirty healthy volunteers were randomly assigned to one of two rtEEG training protocols (SMR?Theta?; Theta?SMR?). Before and after the rtEEG training, participants completed the attention network task (ANT) along with several MW measures. Both training protocols were effective in increasing SMR (SMR?Theta?) and theta (Theta?SMR?) amplitudes but not in decreasing the amplitude of down-trained bands. There were no significant effects of the rtEEG training in either EA or MW measures. However, there was a significant positive correlation between post-training SMR increases and the use of deliberate MW (rather than spontaneous) strategies. Additionally, for the Theta?SMR? protocol, increase in post-training Theta amplitude was significantly associated with a decreased efficiency in the orientation network.     

Cytotoxic copper(II) salicylaldehyde semicarbazone complexes: mode of action and proteomic analysis

The in vitro cytotoxic studies of a series of salicylaldehyde semicarbazones, HOC?H?CH=N-NHCONR? (H?R?) and their Cu(II) complexes on a number of human tumor cell lines were conducted and it was observed that their cytotoxicities were enhanced following complexation to copper. These copper(II) complexes also demonstrated higher in vitro activities than the reference drug, cisplatin, on the tumor cell lines at micro molar range. Apoptotic assays and cell cycle analysis of the copper complexes, [Cu(HBnz?)Cl] and [Cu(HBu?)Cl] revealed that they mediated cytotoxicity in MOLT-4 cells via apoptosis. Further proteomic investigation of [Cu(HBnz?)Cl] and [Cu(HBu?)Cl] with respect to their protein expression profiles associated with their mode of action was conducted. By comparing the expression levels of 33 identified protein spots amongst the respective compound-treated profiles, we identified similarities in protein expression patterns between the two copper(II) complexes. The possible roles of the identified proteins in the execution of apoptosis by these copper(II) complexes are discussed.     

Analysis of functional germline polymorphisms for prediction of response to anthracycline-based neoadjuvant chemotherapy in breast cancer

To elucidate the role of predictive factors on individual's drug response, based on genetic variation, we examined the association between eight germline polymorphisms in genes involved in protection against oxidative stress, apoptosis, oncogenic transformation, proliferation, immune response and DNA repair (TP53, NQO1, IL6, TLR4 and XRCC1) and the pathological response to anthracycline-based neoadjuvant chemotherapy in 70 patients with breast cancer. The DNA was genotyped for eight polymorphisms in five genes (TP53, NQO1, IL6, TLR4 and XRCC1) by 5'-exonuclease (TaqMan?) technology. Fisher's exact test was used to evaluate the association between genotype, clinicopathological parameters and pathological response. A good pathological response, defined as a pathological complete response or residual isolated invasive tumor cells, was found significantly more frequently for estrogen (ER) and progesterone receptor (PR) negative breast carcinomas compared to ER and PR positive and ER or PR positive carcinomas, respectively (43.5 vs. 37.5 and 10.3?%, p?=?0.006), and was significantly associated with high tumor grade (G3) (p?=?0.002). A non-significant trend towards a good pathological response was shown in patients carrying the Arg/Arg or Arg/Pro TP53 codon 72 gene variant compared to those harboring the Pro/Pro variant (17.6 or 37.9?% vs. 0; p?=?0.071). No association was found between NQO1 Pro187Ser, IL6 -174G>C, TLR4 Asp299Gly and Thr399Ile, and XRCC1 Arg194Trp, Arg399Gln and Arg280His and pathological response. The present study shows hormone receptor status and tumor grade as predictors for pathological response to neoadjuvant anthracycline-based chemotherapy. Among various functional germline polymorphisms, a potential predictive value was only found for the TP53 Arg72Pro gene variant.     

L-NAME co-treatment prevent oxidative damage in the lung of adult Wistar rats treated with vitamin A supplementation

Based on the fact that vitamin A in clinical doses is a potent pro-oxidant agent to the lungs, we investigated here the role of nitric oxide (NO?) in the disturbances affecting the lung redox environment in vitamin A-treated rats (retinol palmitate, doses of 1000-9000 IU?kg(-1)?day(-1)) for 28 days. Lung mitochondrial function and redox parameters, such as lipid peroxidation, protein carbonylation and the level of 3-nytrotyrosine, were quantified. We observed, for the first time, that vitamin A supplementation increases the levels of 3-nytrotyrosine in rat lung mitochondria. To determine whether nitric oxide (NO ?) or its derivatives such as peroxynitrite (ONOO-) was involved in this damage, animals were co-treated with the nitric oxide synthase inhibitor L-NAME (30 mg?kg(-1), four times a week), and we analysed if this treatment prevented (or minimized) the biochemical disturbances resulting from vitamin A supplementation. We observed that L-NAME inhibited some effects caused by vitamin A supplementation. Nonetheless, L-NAME was not able to reverse completely the negative effects triggered by vitamin A supplementation, indicating that other factors rather than only NO? or ONOO- exert a prominent role in mediating the redox effects in the lung of rats that received vitamin A supplementation.     

Stimulation of apical Cl⁻/HCO₃⁻(OH⁻) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl?/HCO??(OH?) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl?/HCO??(OH?) exchange activity (～2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl?/HCO??(OH?) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl?/HCO??(OH?) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (～45%) by NPY compared with control. MβCD significantly decreased Cl?/HCO??(OH?) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive 3?Cl? uptake (～50%). Our results demonstrate that NPY modulates Cl?/HCO??(OH?) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl?/HCO??(OH?) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.     

Chromatography and Atomic Absorption Spectrometry for the Assessment of Heavy Metal Distribution among Amino Acids Used in Parenteral Nutrition Formulations—Studies with Cadmium and Lead

The distribution of Cd (II) and Pb (II) among amino acids in parenteral nutrition formulations was investigated by coupling ion-chromatography (HPLC/IC) and electrothermal atomic absorption spectrometry. The methodology was based on ion-exchange separation and fluorimetric amino acid detection after post-column derivatization. Cd (II) and Pb (II) were assayed in 500-µL fractions of the column effluent. The distribution of Cd (II) and Pb (II) in alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His), methionine (Met), phenylalanine (Phe), serine (Ser), and threonine (Thr) were analyzed by monitoring changes in the concentration of free amino acids by HPLC/IC. The results indicated that Cd (II) and Pb (II) were distributed according to the following trend: Gly–Cd?>?Gly–Pb?>?Ala–Cd?>?Ala–Pb?>?His–Cd?～?His–Pb?>?Thr–Cd?>?Thr–Pb?>?Phe–Cd?～?Phe–Pb?～?Asp–Cd?～?Asp–Pb?～?Met–Cd?～?Met–Pb?～?Glu–Cd?～?Glu–Pb?>?Ser–Cd?～?Ser–Pb. The effects of amino acid concentration and stability constants of amino acid–metal complexes are discussed.     

Radiofluorinated rhenium cyclized α-MSH analogues for PET imaging of melanocortin receptor 1

In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several α-melanocyte-stimulating hormone (α-MSH) analogues have been labeled with N-succinimidyl-4-1?F-fluorobenzoate (1?)F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-1?F-fluoropropionate (1?F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma. Metallopeptides Ac-d,Lys-ReCCMSH(Arg11) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with 1?F-NFP or 1?F-NFP. The resulting cold or radiofluorinated metallopeptides, (1?/1?)F-FP-RMSH-1 and (1?/1?)F-FP-RMSH-2, were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution, and small-animal PET imaging properties. The binding affinities of 1?F-FP-RMSH-1 and 1?F-FP-RMSH-2 were determined to be within low nanomolar range. In vivo studies revealed that both F-labeled metallopeptides possessed good tumor uptake in the B16F10 murine model with high MC1R expression, while possessing much lower uptake in A375M human melanoma xenografts. Moreover, 1?F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in the kidney and liver, when compared to that of 1?F-FP-RMSH-2 at 2 h postinjection (p.i.). 1?F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with that of the same peptide labeled with 1?F-SFB (named as 1?F-FB-RMSH-1). Small animal PET imaging of 1?F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organ contrast were observed for A375M model compared to those of the B16F10 model. 1?F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with 1?F-FB-RMSH-1. 1?F-FP-RMSH-1 demonstrates significant advantages over 1?F-FB-RMSH-1 and 1?F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo.     

Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on the N- Methyl-D-Aspartate Receptor lonophore Complex in Rat Brain

Abstract: Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [³H] 5, 7-dichlorokynurenic acid ([³H]- DCKA) but not of the agonist ligand [³H] glycine ([³H] Gly) to a Gly recognition domain on the N -methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [³H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)-α-(4-chlorophenyl)-4- [(4-fluorophenyl)methyl]-1-piperidine ethanol, with [³H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [³H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A₂ and C and p -chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [³H] DCKA binding with [³H] Gly binding being unaltered. Moreover, the densities of [³H] DCKA binding were not significantly different from those of [³H]- Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [³H] Gly binding than of [³H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.     

Activation of protein kinase C beta II by the stereo-specific phosphatidylserine receptor is required for phagocytosis of apoptotic thymocytes by resident murine tissue macrophages

We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AM?) and peritoneal macrophages (PM?) and that such phagocytosis is markedly lower in AM? compared with PM?. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two M? populations. By immunoblotting, AM? expressed equivalent PKC eta but lower amounts of other isoforms (alpha, betaI, betaII, delta, epsilon, mu, and zeta), with the greatest difference in betaII expression. A requirement for PKC betaII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC betaII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC betaII myristoylated peptide as a blocker. Exposure of PM? to phosphatidylserine (PS) liposomes specifically induced translocation of PKC betaII and other isoforms to membranes and cytoskeleton. Both AM? and PM? expressed functional PS receptor, blockade of which inhibited PKC betaII translocation. Our results indicate that murine tissue M? require PKC betaII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in M? phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC betaII activation.     

Doublet translocation at GGA is mediated directly by mutant tRNA(2Gly).

Members of the sufS class of -1 frameshift suppressors have alterations of the GGA/G-decoding tRNA(2Gly). Suppressor-promoted frameshifting at GGA was shown in this study to be directly mediated by the mutant tRNA(2Gly). We disproved the possibility that, in the presence of the compromised mutant tRNA(2Gly), either wild-type tRNA(1Gly), wild-type tRNA(3Gly), a GGA-reading mutant form of tRNA(3Gly), or any other agent suppresses the frameshift mutation trpE91.     

Acute fluoride exposure alters myocardial redox and inflammatory markers in rats

Molecular Biology Reports - Acute fluoride (F?) exposure adversely impairs cardiac functions. We previously reported that acute F? toxicity causes modulation in oxidant and antioxidant...     

Crystallographic and X-ray absorption spectroscopic characterization of Helicobacter pylori UreE bound to Ni²⁺ and Zn²⁺ reveals a role for the disordered C-terminal arm in metal trafficking

The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni2? ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni2? insertion into the apoenzyme. Crystals of apo-HpUreE (H. pylori UreE) and its Ni?- and Zn?-bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 ? (apo), 1.59 ? (Ni2?) and 2.52 ? (Zn2?) resolution, show the conserved proximal and solvent-exposed His1?2 residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apoprotein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His1?2. The analysis of X-ray absorption spectral data obtained using solutions of Ni2?- and Zn2?-bound HpUreE provided accurate information of the metal-ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal-ion binding, and the mutual influence of protein framework and metal-ion stereo-electronic properties in establishing co-ordination number and geometry leading to metal selectivity.     

The influence of the peptide chain length on the activity of peptidyl-tRNA hydrolase from E. coli.

The dependence of the Vmax and Km on the length of the peptide moiety in the peptidyl-tRNA series (Gly)n-Val tRNA, was measured in the system peptidyl-tRNA hydrolase-peptidyl-tRNA. It was found that the Km value decreases from 7.2 X 10-7 M for Gly-Val-tRNA to 4.6 X 10-7 M FOR (Gly)2-Val-tRNA and to 1.7 X 10-7M for (Gly)3-Val-tRNA; further increase of the peptide chain is not followed by decrease of the Km. The Vmax values are 5.7 pmole/min/EU for Gly-Val-tRNA and 42 pmole/min/EU for (Gly)3-Val-tRNA. The enzyme activity is inhibited competitively by uncharged tRNA with a KI value of about 10-5M. The significance of these results described in this paper, in relation to the fact that peptides and peptide esters do not inhibit the enzyme activity, and in relation to the proposed physiological role of the enzyme, is discussed.     

An allosteric modulator of metabotropic glutamate receptors (mGluR₂), (+)-TFMPIP, inhibits restraint stress-induced phasic glutamate release in rat prefrontal cortex

The potential anxiolytic effects of a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor subgroup 2 (mGluR?) were investigated using a self-referencing recording technique with enzyme-based microelectrode arrays (MEAs) that reliably measures tonic and phasic changes in extracellular glutamate levels in awake rats. Studies involved glutamate measures in the rat prefrontal cortex during subcutaneous injections of the following: vehicle, a mGluR?/? agonist, LY354740 (10?mg/kg), or a mGluR? PAM, 1-Methyl-2-((cis-(R,R)-3-methyl-4-(4-trifluoromethoxy-2-fluoro)phenyl)piperidin-1-yl)methyl)-1H-imidazo[4,5-b]pyridine ((+)-TFMPIP; 1.0 or 17.8?mg/kg). Studies assessed changes in tonic glutamate levels and the glutamatergic responses to a 5-min restraint stress. Subcutaneous injection of (+)-TFMPIP at a dose of 1.0?mg/kg (day 3: -7.1?±?15.1 net AUC; day 5: -24.8?±?24.9 net AUC) and 17.8?mg/kg (day 3: -46.5?±?33.0 net AUC; day 5: 34.6?±?36.8 net AUC) significantly attenuated the stress-evoked glutamate release compared to vehicle controls (day 3: 134.7?±?50.6 net AUC; day 5: 286.6?±?104.5 net?AUC), whereas the mGluR?/? agonist LY354740 had no effect. None of the compounds significantly affected resting glutamate levels, which we have recently shown to be extensively derived from neurons. Taken together, these data support that systemic administration of (+)-TFMPIP produces phasic rather than tonic release of glutamate that may play a major role in the effects of stress on glutamate neuronal systems in the prefrontal cortex.     

Bradykinin and prostaglandin E₁ regulate calcitonin gene-related peptide expression in cultured rat sensory neurons

Primary cultures of adult rat dorsal root ganglia (DRG) sensory neurons were used to determine whether bradykinin and prostaglandins E? (PGE?), E? (PGE?) or I? (PGI?) stimulate long-term calcitonin gene-related peptide (CGRP) mRNA accumulation and peptide release. Treatment (24 h) of neurons with either bradykinin or PGE?, significantly increased CGRP mRNA content and iCGRP release. However, PGE? or PGI? was without effect. Exposure of the cultured neurons to increasing concentrations of bradykinin or PGE? demonstrated that the stimulation of CGRP expression was concentration-dependent, while time-course studies showed that maximal levels of CGRP mRNA accumulation and peptide release were maintained for at least 48 h. Treatment of the neuronal cultures with a bradykinin B? receptor antagonist significantly inhibited the bradykinin-induced increase in CGRP expression and release. In addition, preincubation of neuronal cultures with the cyclooxygenase inhibitor indomethacin did not alter the PGE?-mediated stimulation of CGRP but blocked completely the bradykinin-induced increase in CGRP production. Therefore, these data indicate that bradykinin and PGE? can regulate the synthesis and release of CGRP in DRG neurons and that the stimulatory effects of bradykinin on CGRP are mediated by a cyclooxygenase product(s). Thus, these findings suggest a direct relationship between chronic alterations in bradykinin/prostaglandin production that may arise from pathophysiological causes and long-term changes in CGRP expression.     

A 7-kDa prion protein (PrP) fragment, an integral component of the PrP region required for infectivity, is the major amyloid protein in Gerstmann-Sträussler-Scheinker disease A117V

Gerstmann-Str?ussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Str?ussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.     

Toll-like receptor 4 polymorphisms predispose to cutaneous leishmaniasis

The clinical spectrum of cutaneous leishmaniasis (CL) is extremely variable. Studies in experimental leishmaniasis have revealed a role for TLR4 in control of infection. In the present study the associations between TLR4 mutations (Asp299Gly and Thr399Ile) with outcome of CL have been investigated. Genotyping for Asp299Gly and Thr399Ile was performed in patients with chronic (N?=?22) and acute (N?=?61) CL, asymptomatic (N?=?45) and healthy leishmanin skin test negative individuals (N?=?75). The results showed the frequency of the Asp299Gly genotype was increased in patients with chronic disease (OR 25.3, 95% CI 5.2-115.6, P?相似文献