The alpha-subunits of H,K-ATPase (HKAalpha) and Na,K-ATPase require a beta-subunit for maturation. We investigated the role of the beta-subunit in the membrane insertion and stability of the HKAalpha expressed in Xenopus oocytes. Individual membrane segments M1, M2, M3, M4, and M9 linked to a glycosylation reporter act as signal anchor (SA) motifs, and M10 acts as a partial stop transfer motif. In combined HKAalpha constructs, M2 acts as an efficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 and M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined alpha constructs, M1-3 alpha-proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha-proteins are not resistant to cellular degradation. However, co-expression with beta-subunits increases the membrane insertion of M9 in a M1-9 alpha-protein and completely protects M1-10 alpha-proteins against cellular degradation. Our results indicate that HKAalpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular characteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha-subunit depend on intramolecular and intermolecular beta-subunit interactions that are similar but not identical to data obtained for the Na,K-ATPase alpha-subunit.

Myeloperoxidase (MPO) is a hemoprotein that is synthesized in the lumen of the endoplasmic reticulum (ER) as a single-chain precursor and undergoes a complex series of post-translational modifications prior to packaging into azurophilic granules. We and others have previously observed that treatment of human myeloid leukemic cells with succinylacetone (SA), a potent inhibitor of 5-aminolevulinic acid dehydratase (ALA-D), and hence of heme biosynthesis, resulted in loss of MPO enzyme activity, inhibition of the appearance of mature MPO, and accumulation of enzymatically unreactive, but immunoreactive, MPO in the ER. The present study using HL-60 cells was undertaken to establish the nature and specificity of the inhibition by SA and to identify and quantify the biochemical changes in the post-translational pathway of MPO processing. Dose-response studies showed that SA (250 microM) did not affect cell viability or growth up to 72 h, but resulted in inhibition of ALA-D activity >> 93%) and decreased cellular levels of both heme and MPO (approximately 25% of control). There were no effects on the level of total cellular protein or on the activities of lactate dehydrogenase or several other nonheme enzymes colocalized with MPO in azurophilic granules. Northern blot analyses confirmed the nontoxic nature of the conditions and indicated there was no effect on transcription of MPO mRNA. The kinetics of processing in the presence and absence of 250 microM SA were determined using pulse-chase and Percoll density gradient centrifugation methods, followed by identification and quantification of MPO species by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The initial rate of disappearance of precursor MPO was identical for control and SA-treated cells and, after a lag of 2-3 h, there was a fourfold decrease in the rate of appearance of mature MPO in SA-treated cells. In the presence of SA, precursor apoMPO remained in the ER, did not undergo proteolytic processing and, compared to control cells, about 50% was degraded. The disruption in MPO processing was reversible by the addition of exogenous heme. We conclude that the availability of heme is important in the complex maturation of MPO that occurs in the ER, events which precede exit from this compartment and subsequent proteolytic processing and transport to the azurophilic granule.

The sinoatrial (SA) and atrioventricular (AV) nodes are specialized centers of the heart conduction system and are composed of muscle cells with distinctive morphological and electrophysiological properties. We report here results of immunofluorescence and immunoperoxidase studies on the bovine heart showing that a large number of SA and AV nodal cells share a distinct type of myosin heavy chain (MHC) which is not found in other myocardial cells and can thus be used as a cell-type-specific marker. The antibody used in this study was raised against fetal skeletal myosin and reacted with fetal skeletal but not with adult skeletal MHCs. Both atrial and ventricular fibers, as well as fibers of the ventricular conduction tissue were unlabeled by this antibody. Specific reactivity was exclusively seen in most cells in the central portions of the SA and AV nodes and rare cells in perinodal areas. However, a number of nodal cells, particularly those located in the peripheral nodal regions, were unreactive with this antibody. The myosin composition of nodal tissues was also explored using two antibodies reacting specifically with alpha-MHC, the predominant atrial isoform, and beta-MHC, the predominant ventricular isoform. Most nodal cells were reactive for alpha-MHC and a number of them also for beta-MHC. Variation in reactivity with the two antibodies was also observed in perinodal areas: at these sites a population of large fibers reacted exclusively for beta-MHC. These findings point to the existence of muscle cell heterogeneity with respect to myosin composition both in nodal and perinodal tissues.

N5-Acetyl-N5-hydroxy-L-ornithine (1), the key constituent of several microbial siderophores, has been synthesized in 23% yield overall from N-Cbz-L-glutamic acid 1-tert-butyl ester (6) derived from L-glutamic acid. Reduction of 6 to 7 and treatment with N-[(trichloroethoxy)carbonyl]-O-benzylhydroxylamine (8), and diethyl azodicarboxylate and triphenylphosphine followed by deprotection produced the protected N5-acetyl-N5-hydroxy-L-ornithine derivatives 11 and 12 in large quantities (10-20 g). Following alpha-amino and alpha-carboxyl deprotections of 11 and 12, EEDQ [2-ethoxy-N-(ethoxycarbonyl)-1,2-dihydroquinoline] mediated peptide coupling and final deprotection provided amino acid 1 and six albomycin-like peptides (20, 23, 25, 28, 35, and 36). The growth-promoting ability of each was evaluated with the siderophore biosynthesis mutant Shigella flexneri SASA 100 iucD:Tn5). These results indicate that substantial modification of the framework of peptide-based siderophores can be tolerated by microbial iron-transport systems.

Cu(I)-mediated [3+2]cycloaddition between azides and alkynes has evolved into a valuable bioconjugation tool in radiopharmaceutical chemistry. We have developed a simple, convenient and reliable radiosynthesis of 4-[18F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA) as a novel aromatic sulfonamide-based click chemistry building block. [18F]F-SA could be prepared in a remotely controlled synthesis unit in 32 ± 5% decay-corrected radiochemical yield in a total synthesis time of 80 min. The determined lipophilicity of [18F]F-SA (logP = 1.7) allows handling of the radiotracer in aqueous solutions. The versatility of [18F]F-SA as click chemistry building block was demonstrated by the labeling of a model peptide (phosphopeptide), protein (HSA), and oligonucleotide (L-RNA). The obtained radiochemical yields were 77% (phosphopeptide), 55-60% (HSA), and 25% (L-RNA), respectively. Despite the recent emergence of a multitude of highly innovative novel bioconjugation methods for 18F labeling of biopolymers, Cu(I)-mediated click chemistry with [18F]F-SA represents a reliable, robust and efficient radiolabeling technique for peptides, proteins, and oligonucleotides with the short-lived positron emitter 18F.

The Senegalese sole, a high-value flatfish, is a good candidate for aquaculture production. Nevertheless, there are still issues regarding this species' sensitivity to stress in captivity. We aimed to characterize the hepatic proteome expression for this species in response to repeated handling and identify potential molecular markers that indicate a physiological response to chronic stress. Two groups of fish were reared in duplicate for 28 days, one of them weekly exposed to handling stress (including hypoxia) for 3 min, and the other left undisturbed. Two-dimensional electrophoresis enabled the detection of 287 spots significantly affected by repeated handling stress (Wilcoxon-Mann-Whitney U test, p < 0.05), 33 of which could be reliably identified by peptide mass spectrometry. Chronic exposure to stress seems to have affected protein synthesis, folding and turnover (40S ribosomal protein S12, cathepsin B, disulfide-isomerase A3 precursor, cell-division cycle 48, and five distinct heat shock proteins), amino acid metabolism, urea cycle and methylation/folate pathways (methionine adenosyltransferase I α, phenylalanine hydroxylase, mitochondrial agmatinase, serine hydroxymethyltransferase, 3-hydroxyanthranilate 3,4-dioxygenase, and betaine homocysteine methyltransferase), cytoskeletal (40S ribosomal protein SA, α-actin, β-actin, α-tubulin, and cytokeratin K18), aldehyde detoxification (aldehyde dehydrogenase 4A1 family and aldehyde dehydrogenase 7A1 family), carbohydrate metabolism and energy homeostasis (fatty acid-binding protein, enolase 3, enolase 1, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, aconitase 1, mitochondrial ATP synthase α-subunit, and electron-transfer flavoprotein α polypeptide), iron and selenium homeostasis (transferrin and selenium binding protein 1), steroid hormone metabolism (3-oxo-5-β-steroid 4-dehydrogenase), and purine salvage (hypoxanthine phosphoribosyltransferase). Further characterization is required to fully assess the potential of these markers for the monitoring of fish stress response to chronic stressors of aquaculture environment.

The mesoderm of each of the paired lateral heart-forming regions (HFRs) in the stage 5-7 chick embryo includes prospective conus (pre-C), ventricle (pre-V), and sinoatrial (pre-SA) cells, arranged in a rostrocaudal sequence (C-V-SA). With microsurgery we divided each HFR into three rostrocaudally arranged segments. After 24 hr of further incubation, each segment differentiated into a spontaneously beating vesicle of heart tissue to form a multiheart embryo. The cardiac vesicles in these embryos expressed left-right and rostrocaudal beat rate gradients: the left caudal pre-SA mesoderm produced tissue with the fastest beat rate of the six while the rostral vesicle formed from right pre-C was the slowest. In another operation, we prevented the HFRs from fusing in the midline by cutting through the anterior intestinal portal at stage 8, to produce cardia bifida (CB) embryos with an independently beating half-heart on each side. In these cases, the left half-heart of 87.2% of CB embryos beat faster than the right, confirming the left-right difference in intrinsic beat rate. To assess whether the future beat rate of each region is already determined in the st 5-7 HFR, we exchanged rectangular fragments of left pre-SA mesoderm and attached endoderm with right pre-C fragments to yield a left HFR with the sequence C-V-C and a right HFR with the sequence SA-V-SA. A CB operation was subsequently performed on these exchange embryos to prevent fusion of the lateral HFRs. Preconus mesoderm, transplanted to the pre-SA region, differentiated into tissue with a rapid beat rate, while pre-SA mesoderm relocated to the preconus region formed heart tissue with a slow spontaneous rate typical of the conus. In 73% of the exchange CB embryos, the left half-heart beat faster than the right, despite the origins of its mesoderm. The exchanged mesoderm developed a rate that was appropriate for its new location rather than the site of origin of the mesodermal fragment. In a third set of operations, we implanted a fragment of st 15 differentiated conus tissue into a site lateral to the left caudal HFR in st 5, 6, and 7 embryos, and subsequently performed CB operations on them. The implant caused the adjacent half-heart to develop with a slower beat rate than in unoperated or sham-operated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Nicotine patch administration is often used to sustain tobacco abstinence in smoking-cessation programs. There is some concern regarding safety issues, as a consequence of the sympathomimetic action of nicotine. We used spectral analysis of RR interval and (noninvasive) systolic arterial pressure (SAP) beat-by-beat variabilities in a crossover double-blind design to assess the autonomic effects of cigarette smoking, of transdermal nicotine, and of placebo. The study group consisted of 27 heavy smokers (age 43 +/- 2 years). The RR interval and its variability were significantly reduced in the smoking group, as compared with nicotine or placebo groups. The LF component of RR interval variability (in normalized units, nu), and the LF/HF ratio showed greatest values during smoking, as compared with placebo. Values of LF(RR) and LF/HF during nicotine patch treatment were slightly, but not significantly, greater than observed with placebo. No differences were observed in SAP and its variability components. The index alpha (a frequency domain measure of baroreflex gain) was minimal in the smoking period. Habitual cigarette smoking is associated with signs of sympathetic predominance in the autonomic control of the sinoatrial (SA) node. Nicotine patches produce only minor disturbances of autonomic regulation. This corroborates their safe use in smoking-cessation strategies.

The activation of two tobacco MAP kinases, SIPK and WIPK, by a variety of pathogen-associated stimuli and other stresses have been analyzed (Table 1). SIPK was activated by SA, a CWD carbohydrate elicitor and two elicitins from Phytophthora spp, bacterial harpin, TMV, and Avr9 from Cladosporium fulvum. In addition to these pathogen-associated stimuli, wounding also activated SIPK, suggesting that this enzyme is involved in multiple signal transduction pathways. In all cases tested, SIPK activation was exclusively post-translational via tyrosine and threonine/serine phosphorylation. WIPK was activated by only a subset of these stimuli, including infection by TMV or harpin-producing Pseudomonas syringae (preliminary unpubl. result) and treatment with the CWD elicitor, elicitins or Avr9. In contrast to SIPK, WIPK was activated at multiple levels. Low level activation (e.g. by the CWD elicitor) appeared to be primarily post-translational whereas dramatic increases in kinase activity (e.g. by TMV or elicitins) required not only post-translational phosphorylation, but also preceding rises in mRNA levels and de novo synthesis of WIPK protein. Interestingly, under conditions where the same stimulus activated both of these kinases, their kinetics of activation appeared to be distinct. SIPK was the first to be activated. Activation of the low basal level of WIPK protein present before treatment exhibited similar kinetics to that of SIPK; however, the appearance of high levels of WIPK enzyme activity was delayed, perhaps reflecting the need for WIPK transcription and de novo protein synthesis.

Oxidants may play a central role in the pathogenesis of adult respiratory distress syndrome, and phospholipase activation is a potential mechanism of oxidant-induced injury of alveolar epithelial cells. Studies were performed in rat alveolar type II epithelial cells (RAEC) after 3 days in culture. As measured by 51Cr and lactate dehydrogenase release, H2O2 caused time- and dose-dependent cytotoxicity to RAEC. RAEC phospholipids labeled with [14C]-stearic acid ([14C]SA) and [3H]arachidonic acid ([3H]AA) released free fatty acids in response to H2O2 in a manner that closely paralleled the cytotoxicity indexes. Analysis of phospholipid subclasses indicated that phosphatidylcholine was preferentially affected. Analysis for putative products of phospholipase activity revealed significant increases in diacylglycerol and phosphorylcholine, expected products of phospholipase C, as well as significant increases in L-alpha-lysophosphatidylcholine and L-alpha-glycerophosphocholine, expected products of phospholipase A2. Increases in phospholipase D activity were not detected. To determine whether H2O2-stimulated phospholipase activity might be Ca2+ stimulated, RAEC were loaded with fura-2/AM, and changes in intracellular Ca2+ concentrations ([Ca2+]i) were monitored by epifluorescent microscopy. Exposure to H2O2 caused elevations in [Ca2+]i, and the time and dose relationships were consistent with the hypothesis that the release of [14C]SA and [3H]AA is related to changes in cellular Ca2+ concentrations. Additionally, pretreatment with MAPTAM, an intracellular chelator of calcium, partially blocked H2O2-mediated [3H]AA liberation. However, experiments in saponin-permeabilized RAEC, in which [Ca2+]i was strongly buffered by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, indicate that H2O2-induced phospholipase activity also has a Ca(2+)-independent component.(ABSTRACT TRUNCATED AT 250 WORDS)

The nicotinic acetylcholine receptor is an integral membrane protein and a ligand-gated cation channel. It has stoichiometry alpha 2 beta gamma delta, the subunits arranged symmetrically around an approximate five-fold axis. Five M2 helices, one from each subunit, form a parallel helix bundle surrounding a central pore. Simulated annealing via restrained molecular dynamics (SA/MD) has been employed to generate ensembles of isolated M2 transmembrane helices. Four ensembles of two different M2 helix sequences, M2 delta and M2 gamma, have been generated by SA/MD. The ensembles differed in their treatment of electrostatic interactions. Analysis of the simulated structures showed that intra-helical H-bonds were more strongly conserved in the C-terminal (and more hydrophobic) segment of M2 helices. Conformations of polar sidechains have been analyzed, placing particular emphasis on EK (and QK) pairs at the N-termini of M2 delta (and M2 gamma) helices. Conformations of EK sidechain pairs were obtained for the high resolution structures in the protein database in order to guide our analysis of simulated structures. Serine and threonine sidechain conformations in the M2 models also have been determined. Implications of studies of isolated M2 helices for models of the intact pore region of the nicotinic receptor are discussed.

Corticotropin-releasing hormone (CRH) is the principal regulator of the hypothalamic-pituitary-adrenal (HPA) axis and an activator of the sympathoadrenal (SA) and systemic sympathetic (SS) systems. Mental disorders, including major depression and, more recently, Alzheimer's disease have been associated with dysregulation of the HPA axis and the SA/SS systems. Treatment of rats or monkeys with the novel CRH receptor type 1 (CRH-R1) antagonist antalarmin inhibits the HPA and/or the SA/SS axes. This is the first study to examine the potential direct effect of antalarmin on human adrenal function. Adrenocortical and adrenomedullary cells were characterized by double-immunohistochemistry with anti-17 alpha hydroxylase (cortical cells) and anti-chromogranin A (chromaffin cells). Expression of CRH, ACTH, CRH type I and type II receptor mRNA were analyzed by reverse-transcription (RT) PCR. Human adrenal cortical and/or chromaffin cells in co-culture were incubated with CRH, antalarmin, and both CRH and antalarmin in vitro. Exposure of these cells to corticotropin or vehicle medium served as positive and negative controls, respectively. Cortical and chromaffin tissues were interwoven in the human adrenals, and both in situ and in the co-culture system the endocrine cell types were in close cellular contact. ACTH, CRH, and CRH-R1 and CRH-R2 mRNAs were expressed in the human adrenal as determined by RT-PCR. CRH (10-8 M) led to a moderate increase of cortisol release (145.7 +/- 20.0%) from cortical and chromaffin adrenal cells in co-culture. This effect corresponded to 41.8% of the maximal increase induced by ACTH (10-8 M). The action of CRH was completely inhibited by antalarmin. CRH, ACTH, and both CRH-R1 and CRH-R2 mRNAs are expressed in the adult human adrenal gland. CRH stimulates cortisol production in cortical and chromaffin cell co-cultures. This effect is blocked by antalarmin, a selective CRH-R1 receptor antagonist, suggesting that CRH-R1 receptors are involved in an intraadrenal CRH/ACTH control system in humans.

Alpha-momorcharin (α-MMC) is type-1 ribosome inactivating proteins (RIPs) with molecular weight of 29 kDa and has lots of biological activity. Our recent study indicated that the α-MMC purified from seeds of Momordica charantia exhibited distinct antiviral and antifungal activity. Tobacco plants pre-treated with 0.5 mg/mL α-MMC 3 days before inoculation with various viruses showed less-severe symptom and less reactive oxygen species (ROS) accumulation compared to that inoculated with viruses only. Quantitative real-time PCR analysis revealed that the replication levels of viruses were lower in the plants treated with the α-MMC than control plants at 15 days post inoculation. Moreover, the coat protein expression of viruses was almost completely inhibited in plants which were treated with the α-MMC compared with control plants. Furthermore, the SA-responsive defense-related genes including non-expressor of pathogenesis-related genes 1 (NPR1), PR1, PR2 were up-regulated and activities of some antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were increased after the α-MMC treatment. In addition, the α-MMC (500 μg/mL) revealed remarkable antifungal effect against phytopathogenic fungi, in the growth inhibition range 50.35-67.21 %, along with their MIC values ranging from 100 to 500 μg/mL. The α-MMC had also a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of Sclerotinia sclerotiorum. The α-MMC showed a remarkable antiviral and antifungal effect and hence could possibly be exploited in crop protection for controlling certain important plant diseases.

Mitochondrial dysfunction is mainly associated with high-fat-diet (HFD)-induced hepatic steatosis. Sennoside A (SA), a commonly used clinical stimulant laxative, is reported to improve energy metabolism and insulin resistance. However, the effect and mechanism of SA on HFD-induced hepatic steatosis remain largely unknown. The aim of this study was to determine the effect and mechanism of SA on HFD-induced hepatic steatosis in mice. We examined the liver and body weight of mice to evaluate the physical changes in the liver. Hematoxylin and eosin (H&E) and oil red O staining were used to detect the lipid accumulation. The mitochondrial structure and function were tested by transmission electron microscopy and the Seahorse XF24 Analyzer. Furthermore, mitochondrial complexes I, II, and IV and voltage-dependent anion channel 1 (VDAC1) protein activity were detected to understand the mechanism of the protective effect on mitochondria. As a result, damage to the structure and function in the hepatic mitochondria of HFD-induced hepatic steatosis was observed in mice. The structural damage was in the form of loss of cristae, mitochondrial swelling, vacuolization and even rupturing of the outer mitochondrial membrane (OMM). Functional alterations were found by activation of complex I and deficiency in complexes II and IV. The VDAC1 activity and the total ATP in the liver tissue was increased under hepatic steatosis conditions. The above effects were reversed by SA. These data suggest that inhibition of VDAC1 may be an underlying mechanism of SA for protecting mitochondria in HFD-induced hepatic steatosis in mice. Thus, VDAC1 may be a promising target for treating fatty liver disease.

Salicylic acid (SA) plays a critical role in plant defense against pathogen invasion. SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense and involves a specific pathway mediated by mitochondria; however, the underlying mechanisms remain largely unknown. The SA-binding activity of the recombinant tomato (Solanum lycopersicum) alpha-ketoglutarate dehydrogenase (Slα-kGDH) E2 subunit of the tricarboxylic acid (TCA) cycle was characterized. The biological role of this binding in plant defenses against tobacco mosaic virus (TMV) was further investigated via Slα-kGDH E2 silencing and transient overexpression in plants. Slα-kGDH E2 was found to bind SA in two independent assays. SA treatment, as well as Slα-kGDH E2 silencing, increased resistance to TMV. SA did not further enhance TMV defense in Slα-kGDH E2-silenced tomato plants but did reduce TMV susceptibility in Nicotiana benthamiana plants transiently overexpressing Slα-kGDH E2. Furthermore, Slα-kGDH E2-silencing-induced TMV resistance was fully blocked by bongkrekic acid application and alternative oxidase 1a silencing. These results indicated that binding by Slα-kGDH E2 of SA acts upstream of and affects the mitochondrial electron transport chain, which plays an important role in basal defense against TMV. The findings of this study help to elucidate the mechanisms of SA-induced viral defense.

Two monoclonal antibodies, TKH2 and B72.3, directed toward the Sialyl-Tn antigen (SA 2, 6GalNAc alpha-O-Ser/Thr), were examined immunohistochemically to analyze the expression of these antigens in 20 areas of normal squamous epithelium, 12 lesions of dysplasia, and 86 cases of squamous cell carcinoma including 32 with superficial carcinoma in the esophagus. No expression of TKH2 or B72.3 was found in the normal squamous epithelium. Among the 12 lesions of dysplasia only one expressed TKH2. In carcinoma the expression of TKH2 and B72.3 was found in 40 (47%) and 21 (24%) of the 86 carcinomas, respectively; however, the number of positive malignant cells with TKH2 and B72.3 totaled less than half that in the tissue, and no relationship was found between either prognosis or lymph node metastasis and the expression of Sialyl-Tn antigen. These results indicate that Sialyl-Tn antigen appears in the process of malignant transformation or tumor progression in esophageal squamous cell carcinoma; however, the positive expression of Sialyl-Tn antigen was not directly connected to either prognosis or lymph node metastasis.

Background: Liver fibrosis occurs due to chronic liver disease due to multiple pathophysiological causes. The main causes for this condition are chronic alcohol abuse, nonalcoholic steatohepatitis, and infection due to hepatitis C virus. Currently, there is more and more information available about the molecular as well as cellular mechanisms, which play a role in the advancement of liver fibrosis. However, there is still no effective therapy against it. Purpose: In order to find an effective treatment against liver fibrosis, our study explored whether salvianolic acid A (SA-A), a traditional Chinese medicine extracted from the plant Danshen, could effectively inhibit the liver fibrosis, which is induced by CCl₄ in vivo. Methods: The effects of SA-A were evaluated by assessing the parameters related to liver fibrosis such as body weight, histological changes, and biochemical parameters. Thereafter, the related protein or gene levels of P13K/AKT/mTOR, Bcl-2/Bax and caspase-3/cleaved caspase-3 signaling pathways were determined by western blotting, real-time PCR or immunohistochemistry staining. Results: According to the results of our study, SA-A could reduce liver fibrosis by inhibiting liver function, liver fibrosis index, collagen deposition, and improving the degree of liver fibrosis in rats. Mechanistically, the PI3K/AKT/mTOR signaling cascade was inhibited by SA-A to prevent the stimulation of hepatic stellate cell, as well as the synthesis of extracellular matrix, and regulated Bcl-2/Bax and caspase-3/cleaved caspase-3 signaling pathways to prevent hepatocyte apoptosis. Conclusion: The novel findings of this study suggested that SA-A could reduce liver fibrosis and the molecular mechanisms behind it are closely associated with the regulation of PI3K/AKT/mTOR, Bcl-2/Bax and caspase-3/cleaved caspase-3 signaling pathways.

OBJECTIVE

We used acetylsalicylic acid (ASA) as a probing agent to quantify hydroxyl radical ((*)OH) in Controls and patients with coronary artery disease and to prospectively investigate (*)OH production in patients with myocardial infarction (MI) complicated by heart failure (HF).

BACKGROUND

Oxidative stress status (OSS) is a mechanism for transition to HF in experimental heart injury models, but evidence for its causal role in humans is still limited.

METHODS

Thirty healthy subjects (Controls), 12 patients with stable angina (Group 1), and 74 patients with ST-segment elevation MI (Group 2) were enrolled. A dose of 250 mg Flectadol was given intravenously before each blood collection to determine the 2,3-dihydroxybenzoic acid/salicylic acid (DHBA/SA) ratio. We also quantified vitamin E and coenzyme Q(10) to monitor antioxidant reserve, as well as tumor necrosis factor (TNF)-alpha, TNF-soluble receptors, interleukin (IL)-6, and IL-1ra to assess inflammatory status. All measurements were repeated at month 6 in Group 2.

RESULTS

There were no differences between Controls and Group 1. Group 2 showed increased (*)OH production, peaking at 24 h, whereas vitamin E and coenzyme Q(10) progressively declined. Group 2 patients developing HF during hospitalization (Group 2Bi) presented with an increase of both (*)OH production at discharge and inflammatory status, as compared with patients without HF (Group 2Ai), persisting at month 6 in post-MI patients with HF (Group 2Bii).

CONCLUSIONS

We found a distinct pattern of (*)OH generation in post-MI patients who show progression to HF. The interplay between OSS and inflammatory status should be targeted as a possible mechanism of progression to post-MI left ventricular dysfunction.

Various species of fungi in the genus Aspergillus are the most common causative agents of invasive aspergillosis and/or producers of hepato-carcinogenic mycotoxins. Salicylaldehyde (SA), a volatile natural compound, exhibited potent antifungal and anti-mycotoxigenic activities to A. flavus and A. parasiticus. By exposure to the volatilized SA, the growth of A. parasiticus was inhibited up to 10-75% at 9.5 mM ≤ SA ≤ 16.0 mM, while complete growth inhibition was achieved at 19.0 mM ≤ SA. Similar trends were also observed with A. flavus. The aflatoxin production, i.e., aflatoxin B(1) and B(2) (AFB(1), AFB(2)) for A. flavus and AFB(1), AFB(2), AFG(1), and AFG(2) for A. parasiticus, in the SA-treated (9.5 mM) fungi was reduced by ~13-45% compared with the untreated control. Using gene deletion mutants of the model yeast Saccharomyces cerevisiae, we identified the fungal antioxidation system as the molecular target of SA, where sod1Δ [cytosolic superoxide dismutase (SOD)], sod2Δ (mitochondrial SOD), and glr1Δ (glutathione reductase) mutants showed increased sensitivity to this compound. Also sensitive was the gene deletion mutant, vph2Δ, for the vacuolar ATPase assembly protein, suggesting vacuolar detoxification plays an important role for fungal tolerance to SA. In chemosensitization experiments, co-application of SA with either antimycin A or strobilurin (inhibitors of mitochondrial respiration) resulted in complete growth inhibition of Aspergillus at much lower dose treatment of either agent, alone. Therefore, SA can enhance antifungal activity of commercial antifungal agents required to achieve effective control. SA is a potent antifungal and anti-aflatoxigenic volatile that may have some practical application as a fumigant.

Previously it was shown that combined low dose treatment of tocotrienols and statins synergistically inhibited the growth of highly malignant +SA mammary epithelial cells in culture. Therefore, the objective of the present work was to prepare and characterize lipid nanoparticles that combined simvastatin and tocotrienol rich fraction (TRF) as potential anticancer therapy. The entrapment of simvastatin in the oily nanocompartments, which were formed by TRF inclusion into the solid matrix of the nanoparticles, was verified by its high entrapment efficiency and the absence of endothermic or crystalline peaks when blends were analyzed by DSC and PXRD, respectively. The release of simvastatin from the nanoparticles in sink conditions was characterized by an initial burst release of approximately 20% in 10h followed by a plateau. No significant change in particle size (approximately 100 nm) was observed after storage for six months. The anticancer activity of the nanoparticles was verified in vitro by observing their antiproliferative effects on malignant +SA mammary epithelial cells. The IC(50) of the reference alpha-tocopherol nanoparticles was 17.7 microM whereas the IC(50) of the simvastatin/TRF nanoparticles was 0.52 microM, which confirmed the potency of the combined treatment and its potential in cancer therapy.

BACKGROUND

Controversy exists concerning the use of fiber-reinforced posts to improve bond strength to resin cement because some precementation treatments can compromise the mechanical properties of the posts.

OBJECTIVE

The purpose of this study was to analyze the influence of airborne-particle abrasion on the mechanical properties and microtensile bond strength (MTBS) of carbon/epoxy and glass/bis-GMA fiber-reinforced resin posts.

METHODS

Flexural strength (delta(f)), flexural modulus (E(f)), and stiffness (S) were assessed using a 3-point bending test for glass fiber-reinforced and carbon fiber-reinforced resin posts submitted to airborne-particle abrasion (AB) with 50-microm Al(2)O(3), and for posts without any surface treatment (controls) (n=10). Forty glass fiber (GF) and 40 carbon fiber (CF) posts were submitted to 1 of 4 surface treatments (n=10) prior to MTBS testing: silane (S); silane and adhesive (SA); airborne-particle abrasion with 50-microm Al(2)O(3) and silane (ABS); airborne-particle abrasion, silane, and adhesive (ABSA). Two composite resin restorations (Filtek Z250) with rounded depressions in the lateral face were bilaterally fixed to the post with resin cement (RelyX ARC). Next, the specimen was sectioned with a precision saw running perpendicular to the bonded surface to obtain 10 bonded beam specimens with a cross-sectional area of 1 mm(2). Each beam specimen was tested in a mechanical testing machine (EMIC 2,000 DL), under stress, at a crosshead speed of 0.5 mm/min until failure. Data were analyzed by 2-way ANOVA followed by Tukey HSD test (alpha=.05). Failure patterns of tested specimens were analyzed using scanning electron microscopy (SEM).

RESULTS

The 3-point bending test demonstrated significant differences among groups only for the post type factor for flexural strength, flexural modulus, and stiffness. The carbon fiber posts exhibited significantly higher mean flexural strength (P=.001), flexural modulus (P=.003), and stiffness (P=.001) values when compared with glass fiber posts, irrespective of surface treatment. An alteration in the superficial structure of the posts could be observed by SEM after airborne-particle abrasion. MTBS testing showed no significant effect for the surface treatment type; however, significant effects for post system factor and for interaction between the 2 factors were observed. For the carbon fiber post, the ABSA surface treatment resulted in values significantly lower than the S surface treatment. SEM analysis of MTBS-tested specimens demonstrated adhesive and cohesive failures.

CONCLUSIONS

Airborne-particle abrasion did not influence the mechanical properties of the post; however, it produced undesirable surface changes, which could reduce the bond strength to resin cement. For the surface treatments studied, if silane is applied, the adhesive system and airborne-particle abrasion are not necessary.

We previously reported that the mouse alpha/beta hydrolase domain containing 2 (Abhd2) was expressed in smooth muscle cells (SMCs) which suppressed their migration and inhibited the development of intimal hyperplasia by cuff placement; however, the role of ABHD2 in human remains to be elucidated. In this study, we examined ABHD2 expression in the human coronary atherosclerotic lesions of the patients with unstable angina (UA) and stable angina (SA). Our results showed that the ABHD2 was expressed in atherosclerotic lesions, and that the ABHD2 expression was significantly higher in the patients with UA than with SA. Immunohistochemistry analysis revealed abundant expression of ABHD2 in macrophages, but low expression in SMCs of atherosclerotic lesions. Using human vascular primary culture cell lines, we also demonstrated that the expression of ABHD2 was significantly higher in macrophages than in SMCs, and that the expression of ABHD2 significantly increased proportionally with differentiation from monocyte into macrophage.

The objective of the present study was to investigate the effects of syringic acid (SA), a phenolic acid, on N(ω)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Hypertension was induced in adult male albino rats by oral administration of L-NAME (40 mg/kg/day) dissolved in drinking water daily for 4 weeks. Rats were treated with different doses of SA (25, 50, and 100 mg/kg body weight (b.w.)). Systolic blood pressure of control and experimental rats was recorded. Plasma nitric oxide metabolites (NOx), lipid peroxidative products such as thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes, and antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, vitamin C, vitamin E, and reduced glutathione were estimated in erythrocytes, plasma, and tissues of experimental rats. Hepatic marker enzymes such as aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase and renal functional markers such as urea, uric acid, and creatinine were also estimated in serum. The increased levels of blood pressure, lipid peroxidation products, hepatic and renal function markers, and the decreased level of NOx and antioxidants in L-NAME-induced hypertensive rats were reversed upon SA treatment. The protective effect at the dose of the three tested doses (25, 50, and 100 mg/kg) of SA at a dose of 50 mg/kg b.w. exerts optimum protection. Biochemical findings are substantiated by the histological observation. The protective effects of SA are mediated by reducing oxidative stress and retaining the bioavailability of NO in the cardiovascular system.

Pancreatic cancer is one of the most malignant tumors. Invasion and metastasis can occur in the early stage of pancreatic cancer, contributing to the poor prognosis. Accordingly, in this study, we evaluated the molecular mechanisms underlying invasion and metastasis. Using mass spectrometry, we found that Integrin alpha 6 (ITGA6) was more highly expressed in a highly invasive pancreatic cancer cell line (PC-1.0) than in a less invasive cell line (PC-1). Through in vitro and in vivo experiments, we observed significant decreases in invasion and metastasis in pancreatic cancer cells after inhibiting ITGA6. Based on data in TCGA, high ITGA6 expression significantly predicted poor prognosis. By using Co-IP combined mass spectrometry, we found that ribosomal protein SA (RPSA), which was also highly expressed in PC-1.0, interacted with ITGA6. Similar to ITGA6, high RPSA expression promoted invasion and metastasis and indicated poor prognosis. Interestingly, although ITGA6 and RPSA interacted, they did not mutually regulate each other. ITGA6 and RPSA affected invasion and metastasis via the PI3K and MAPK signaling pathways, respectively. Inhibiting ITGA6 significantly reduced the expression of p-AKT, while inhibiting RPSA led to the downregulation of p-ERK1/2. Compared with the inhibition of ITGA6 or RPSA alone, the downregulation of both ITGA6 and RPSA weakened invasion and metastasis to a greater extent and led to the simultaneous downregulation of p-AKT and p-ERK1/2. Our research indicates that the development of drugs targeting both ITGA6 and RPSA may be an effective strategy for the treatment of pancreatic cancer.