BACKGROUND/AIMS: Chronic cholestasis stimulates a fibroductular reaction which may progress to secondary biliary fibrosis and cirrhosis. Since platelet-derived growth factor has been indicated as a major fibrogenic factor in chronic liver disease, we analyzed its expression and that of its receptor
beta subunit in a rat model of chronic cholestasis. METHODS: Liver tissue samples collected at 7, 10, 21, and 28 days after induction of cholestasis obtained by bile duct ligation, were analyzed by immunohistochemistry, in situ hybridization and RNase protection assay for the expression of platelet-derived growth factor (PDGF)-B chain and receptor beta subunit. Furthermore, the expression of PDGF-B chain mRNA was analyzed in highly purified cholangiocytes from normal and cholestatic rat liver. RESULTS: In cholestatic liver, platelet-derived growth factor-BB and B chain mRNA expression increased up to 4 weeks in epithelial cells of proliferating bile ducts, and periductular mesenchymal cells. The increased expression of PDGF-B chain mRNA was confirmed in highly purified cholangiocytes obtained from normal and cholestatic rat liver. The expression of the receptor beta subunit progressively increased after induction of cholestasis and was mainly localized to desmin-positive periductular hepatic stellate cells. CONCLUSIONS: These data suggest that platelet-derived growth factor-B chain can be synthesized by cholangiocytes during chronic cholestasis. The presence of its receptor on periductular hepatic stellate cells raises the possibility that, in this experimental setting, this cytokine might contribute to fibrogenesis in vivo.
The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited lysosomal storage diseases and the prototype of childhood onset neurodegenerative disorders. To date, 10 NCL entities (CLN1-CLN10) are known and characterized by accumulation of autofluorescent storage material, age of onset and clin
ical symptoms. CLN8 was first identified as the causative gene for a late-onset form with progressive epilepsy and mental retardation in Finnish patients. In addition, CLN8 phenotypes were described in Turkish, Israeli and Italian patients with a more rapid progression of visual loss, epilepsy, ataxia and mental decline. Here, we report the first mutations in German (c.611G>T) and Pakistani (c.709G>A) patients. Our findings confirm previous assumptions that the CLN8 variant can occur in many ethnic groups. So far, large CLN gene deletions are only known for the CLN3 gene. Here, we also describe a novel, large CLN8 gene deletion c.544-2566_590del2613 in a Turkish family with a slightly more severe phenotype. Our data indicate that patients with clinical signs of late infantile NCL and characteristic ultrastructural inclusions should also be screened for CLN8 mutations independent of their ethnic origin.
Pal A, etal., J Biol Chem. 2009 Feb 6;284(6):3976-84. doi: 10.1074/jbc.M806947200. Epub 2008 Nov 26.
Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We cr
ystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 angstroms resolution reveals a globular structure with a subtilisin-like fold, a Ser475-Glu272-Asp360 catalytic triad, and an octahedrally coordinated Ca2+-binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.
Decay accelerating factor (DAF), a key complement activation control protein, is a 70 kDa membrane bound glycoprotein which controls extent of formation of the C3 and C5 convertases by accelerating their decay. Using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated prot
ein 9 (Cas9) genome editing we generated a novel DAF deficient (Daf-/-) rat model. The present study describes the renal and extrarenal phenotype of this model and assesses renal response to complement-dependent injury induced by administration of a complement-fixing antibody (anti-Fx1A) against the glomerular epithelial cell (podocyte). Rats generated were healthy, viable and able to reproduce normally. Complete absence of DAF was documented in renal as well as extra-renal tissues at both protein and mRNA level compared to Daf+/+ rats. Renal histology in Daf-/- rats showed no differences regarding glomerular or tubulointerstitial pathology compared to Daf+/+ rats. Moreover, there was no difference in urine protein excretion (ratio of urine albumin to creatinine) or in serum creatinine and urea levels. In Daf-/- rats, proteinuria was significantly increased following binding of anti-Fx1A antibody to podocytes while increased C3b deposition was observed. The DAF knock-out rat model developed validates the role of this complement cascade regulator in immune-mediated podocyte injury. Given the increasing role of dysregulated complement activation in various forms of kidney disease and the fact that the rat is the preferred animal for renal pathophysiology studies, the rat DAF deficient model may serve as a useful tool to study the role of this complement activation regulator in complement-dependent forms of kidney injury.
In recent years, there is growing research interest for the biological role of adipose tissue-derived bioactive factors, mainly including adipokines, in various forms of cancer. Adiponectin (APN) is the most abundant circulating adipokine, and a key mediator of several cancer-related processes, such
as cell proliferation, apoptosis, regulation of tumor cell invasion and angiogenesis. In this review we summarize and critically discuss the published literature on the diverse roles of APN in non-small-cell lung cancer, including its implication in lung cancer development, its use as a diagnostic and prognostic biomarker, and its correlation with cancer-related cachexia. The main challenges and future perspectives, mainly with regard to the potential development of APN-targeted therapeutic agents in cancer therapeutics, are also briefly presented and discussed.
Athanassiadou P, etal., Pathol Res Pract. 2008;204(4):241-9. Epub 2008 Jan 2.
We investigated the prognostic significance of cyclooxygenase-2 (COX-2) and survivin in ovarian carcinoma. Imprint smears were obtained from 100 ovarian carcinoma specimens and were studied immunocytochemically for the expression of COX-2 and survivin. The results were correlated with several clinic
opathological parameters, including 5-year survival. Increased COX-2 staining pattern correlated with a non-mucinous histological type (p=0.008), increased stage (p<0.0001), high histological grade (p<0.0001), and reduced survival rates (p<0.00001). Survivin expression was strongly associated with increased stage (p<0.0001), increased histological grade (p<0.0001), and reduced survival (p<0.00001). Elevated survivin expression also correlated significantly with pre-menopausal status (p=0.033). In addition, COX-2 and survivin staining patterns correlated strongly with one another (p<0.0001). However, on multivariate analysis, an independent prognostic value was found only for tumor stage and grade. The findings of our study indicate that the increased expression of COX-2 and survivin in ovarian cancer is associated with one another and with several adverse clinicopathologic parameters, including reduced survival, thus suggesting a role of these molecules in disease progression. Further investigations of the exact prognostic and therapeutic implications of COX-2 and survivin expression are strongly warranted.
Coulon S, etal., Nat Med. 2011 Oct 23;17(11):1456-65. doi: 10.1038/nm.2462.
Anemia because of insufficient production of and/or response to erythropoietin (Epo) is a major complication of chronic kidney disease and cancer. The mechanisms modulating the sensitivity of erythroblasts to Epo remain poorly understood. We show that, when cultured with Epo at suboptimal concentrat
ions, the growth and clonogenic potential of erythroblasts was rescued by transferrin receptor 1 (TfR1)-bound polymeric IgA1 (pIgA1). Under homeostatic conditions, erythroblast numbers were increased in mice expressing human IgA1 compared to control mice. Hypoxic stress of these mice led to increased amounts of pIgA1 and erythroblast expansion. Expression of human IgA1 or treatment of wild-type mice with the TfR1 ligands pIgA1 or iron-loaded transferrin (Fe-Tf) accelerated recovery from acute anemia. TfR1 engagement by either pIgA1 or Fe-Tf increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These cellular responses were mediated through the TfR1-internalization motif, YXXPhi. Our results show that pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. Targeting this pathway may provide alternate approaches to the treatment of ineffective erythropoiesis and anemia.
Pinzani M, etal., Hepatology. 1994 Mar;19(3):701-7.
Platelet-derived growth factor has been shown to play an important role in the repair process after acute tissue injury and in the pathogenesis of several fibrogenic disorders. The aim of this study was to evaluate whether increased expression of platelet-derived growth factor and its beta-receptor
subunit occurs in a model of acute liver injury. Male Sprague-Dawley rats were given a single intragastric dose of carbon tetrachloride and killed at intervals of 24, 48 and 72 hr and 1 wk. Control animals were included in each group. Platelet-derived growth factor-B chain mRNA expression, analyzed by RNase protection assay, was not detectable in control samples or in samples obtained 24 hr or 1 wk after carbon tetrachloride. However, the presence of protected fragments of 130 kb was clearly detected after 48 hr and was still present, although less abundant, after 72 hr. The distribution of platelet-derived growth factor protein in liver tissue sections, evaluated by immunohistochemistry, was restricted to centrilobular veins and portal tracts in normal liver. In carbon tetrachloride-treated rats, prominent staining was observed in areas corresponding to hepatocellular necrosis and inflammatory infiltration. This feature, already present at 24 hr after carbon tetrachloride, became more marked at 48 hr with a gradual resolution after 72 hr. The expression of platelet-derived growth factor-receptor beta-subunit mRNA, evaluated by in situ hybridization, was markedly increased after carbon tetrachloride with a peak at 24 hr and was mainly localized over mesenchymal cells in the hepatic sinusoids.(ABSTRACT TRUNCATED AT 250 WORDS)
Calabro A, etal., Digestion. 2004;70(2):127-38. Epub 2004 Sep 16.
BACKGROUND/AIM: Controlled proteolysis is a prerequisite for cell migration, angiogenesis, and matrix remodelling during gastric ulcer healing. We studied the temporal and spatial expression of three matrix metalloproteinases, gelatinase A (MMP-2), interstitial collagenase (MMP-13), stromelysin (MM
P-3), and their major inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) during experimental gastric ulcer healing induced in rats by acetic acid injection. METHODS: Gastric tissue specimens were hybridized with antisense (35)S-labelled RNA probes and the autoradiographic signal was analyzed by a computer aided image system. Gelatinase activity was analyzed by in situ and gel zymography. RESULTS: During gastric ulcer healing, MMP-2, MMP-3, and MMP-13 RNA expression was increased in stromal cells of the gastric mucosa bordering the ulcer, suggesting a prevalent role of non-epithelial cells in pericellular proteolysis. Gelatinolytic activity was increased during ulcer healing and it was associated with extracellular matrix of the healing mucosa and newly formed vessels. In contrast to MMP-2 RNA, which was homogeneously distributed in all layers of the ulcer bed, MMP-3 and MMP-13 RNAs were confined to the upper layers of the granulation tissue. TIMP-1 RNA was detected in both epithelial and stromal cells of the gastric mucosa adjacent to the ulcer, as well as in the granulation tissue of the ulcer bed. Both MMP and TIMP-1 expression returned to basal levels during the late stages of tissue remodeling. CONCLUSION: Gastric ulcer repair is associated with a transient expression of specific metalloproteinases and their inhibitors in a distinct anatomical pattern pointing to complex cellular and cell/matrix interactions in the various layers of the healing mucosa.
Palagano E, etal., J Bone Miner Res. 2015 Oct;30(10):1814-21. doi: 10.1002/jbmr.2517. Epub 2015 May 21.
Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exom
e sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes.
AIMS/HYPOTHESIS: It is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruct
ion and associated alterations of islet vascularisation. METHODS: NOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation. RESULTS: We demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin-zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet. CONCLUSIONS/INTERPRETATION: FOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted human islets. These results are contributing to a deeper understanding of human islet transplant rejection and label-free in vivo monitoring of drug efficacy.
Li C, etal., Proc Natl Acad Sci U S A. 2019 Jan 22;116(4):1347-1352. doi: 10.1073/pnas.1810951116. Epub 2019 Jan 4.
We have identified a GRAP variant (c.311A>T; p.Gln104Leu) cosegregating with autosomal recessive nonsyndromic deafness in two unrelated families. GRAP encodes a member of the highly conserved growth factor receptor-bound pro
tein 2 (GRB2)/Sem-5/drk family of proteins, which are involved in Ras signaling; however, the function of the growth factor receptor-bound protein 2 (GRB2)-related adaptor protein (GRAP) in the auditory system is not known. Here, we show that, in mouse, Grap is expressed in the inner ear and the protein localizes to the neuronal fibers innervating cochlear and utricular auditory hair cells. Downstream of receptor kinase (drk), the Drosophila homolog of human GRAP, is expressed in Johnston's organ (JO), the fly hearing organ, and the loss of drk in JO causes scolopidium abnormalities. drk mutant flies present deficits in negative geotaxis behavior, which can be suppressed by human wild-type but not mutant GRAP. Furthermore, drk specifically colocalizes with synapsin at synapses, suggesting a potential role of such adaptor proteins in regulating actin cytoskeleton dynamics in the nervous system. Our findings establish a causative link between GRAP mutation and nonsyndromic deafness and suggest a function of GRAP/drk in hearing.
Systemic and local signals must be integrated by mammary stem and progenitor cells to regulate their cyclic growth and turnover in the adult gland. Here, we show RANK-positive luminal progenitors exhibiting WNT pathway activation are selectively expanded in the human breast during the progesterone-h
igh menstrual phase. To investigate underlying mechanisms, we examined mouse models and found that loss of RANK prevents the proliferation of hormone receptor-negative luminal mammary progenitors and basal cells, an accompanying loss of WNT activation, and, hence, a suppression of lobuloalveologenesis. We also show that R-spondin1 is depleted in RANK-null progenitors, and that its exogenous administration rescues key aspects of RANK deficiency by reinstating a WNT response and mammary cell expansion. Our findings point to a novel role of RANK in dictating WNT responsiveness to mediate hormone-induced changes in the growth dynamics of adult mammary cells.
Chen Y, etal., Sci Transl Med. 2015 Dec 9;7(317):317ra197. doi: 10.1126/scitranslmed.aad0295.
Osteosarcoma (OS) is the most common primary bone cancer, which occurs primarily in children and adolescents, severely affecting survivors' quality of life. Despite its chemosensitivity and treatment advances, long-term survival rates for OS patients have stagnated over the last 20 years. Thus, it
is necessary to develop new molecularly targeted therapies for this metastatic bone cancer. Mutations in TP53 and RB are linked to OS predisposition and to the evolution of spontaneous OS. We established receptor activator of nuclear factor kappaB ligand (RANKL) as a therapeutic target for suppression and prevention of OS. Combined conditional osteoblast-specific deletions of Rb, p53, and the protein kinase A (PKA) regulatory subunit Prkar1alpha genes in genetically engineered mouse models (GEMMs) generate aggressive osteosarcomas, characterized by PKA, RANKL, and osteoclast hyperactivity. Whole-body Rankl deletion completely abrogates tumorigenesis. Although osteoblastic Rank deletion has little effect, osteoclastic Rank deletion delays tumorigenesis and prolongs life span. The latter is associated with inactivation of osteoclastogenesis and up-regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Further, we use these GEMMs as preclinical platforms to show that RANKL blockade with RANK-Fc arrests tumor progression and improves survival and also inhibits lung metastasis. Moreover, preemptive administration of RANK-Fc completely prevents tumorigenesis in mice highly predisposed to this aggressive cancer. Denosumab, a fully human monoclonal antibody against RANKL, is currently used to treat patients with osteoporosis or bone metastases. Our studies provide a strong rationale to consider RANKL blockade for the treatment and prevention of aggressive RANKL-overexpressing OS in humans.
Mahmoudian M J Mol Graph. 1994 Mar;12(1):22-8, 34.
Three-dimensional (3D) models of the human Gs protein, the human beta 3 adrenergic receptor and their complex are constructed using computer-aided molecular modeling techniques. The structures of bacterial EF-tu 200 and bacteriorhodopsin were used as starting points for modeling the Gs protein and b
eta 3 receptor, respectively. Experimental data are used as constraints to guide the modeling. The resulting 3D structures of the Gs protein, the beta 3 receptor and their complex are in accord with the experimental data. It is found that the third intracellular loop of the beta 3 receptor as well as its C-terminus are involved in the binding. Various residues of N-terminus and C-terminus of the Gs protein also participate in the binding. The model of the complex suggests that the Gs protein binds to the beta 3 receptor in such a way that it will be placed in the interface of membrane and intracellular space. This orientation is supported by experimental data. It is concluded that the modeled structure of the complex of the alpha subunit of the human Gs protein and the beta 3 adrenergic receptor is in agreement with the experimental data and it can provide a basis for understanding the way these proteins interact.
Jinkins JR Comput Med Imaging Graph 1991 Sep-Oct;15(5):323-31.
Since the original observations of cerebral disconnection in experimental conditions with animal models, the surgical division of the cerebral commissures for control of seizure activity has led to the description of a human hemispheric disconnection syndrome. More recently, MR has revealed a spectr
um of conditions of the forebrain responsible for the natural development and acquired occurrence of this unique commissural syndrome. Acutely, nonspecific expressions of disconnection may be observed; however, there are no explicit behavioral manifestations of this syndrome in the chronic state. The signs of stabilized hemispheric disconnection are only elicitable via sophisticated, neuropsychological testing performed by experienced technicians. Included in the present series of callosal pathology were individuals with agenesis, surgical section, infarction, trauma, glioma, and Marchiafava-Bignami disease. On specialized examination, the surgical "split-brain" patients illustrated the classic syndrome of a halt of interhemispheric transfer of information, a block of nondominant hemisphere access to language functions and an inhibition of dominant hemisphere access to superior visuospatial capacity. The nonsurgical subjects with developmental callosal agenesis and acquired pathologic processes involving the callosum revealed a varied, nonspecific reduction in cognitive function most probably related to associated extracallosal hemispheric pathology. No external abnormalities in behavior which could be attributed specifically to the callosal findings were identified chronically, although some progressive diseases suggested the presence of a subacute symptomatic phase of the disconnection syndrome. Any condition which causes the division, destructive insult, or primary ontogenic nondecussation of commissural axons will produce an interference in interhemispheric, interneuronal communication. This is manifested by individual degrees of covert interhemispheric data transfer arrest and of reduced bidirectional dominant-nondominant hemispheric access and telencephalic integrative capacity, consistent with a cerebral commissuropathy. This study reveals the MR equivalents of the hemispheric disconnection syndrome which occur in many nonspecific pathologic conditions of the cerebrum, but which result in few if any overt behavioral aberrations in the stabilized state.
Post hoc assignment of patterns determined by all pairwise comparisons in microarray experiments with multiple treatments has been proven to be useful in assessing treatment effects. We propose the usage of transitive directed acyclic graphs (tDAG) as the repres
entation of these patterns and show that such representation can be useful in clustering treatment effects, annotating existing clustering methods, and analyzing sample sizes. Advantages of this approach include: (1) unique and descriptive meaning of each cluster in terms of how genes respond to all pairs of treatments; (2) insensitivity of the observed patterns to the number of genes analyzed; and (3) a combinatorial perspective to address the sample size problem by observing the rate of contractible tDAG as the number of replicates increases. The advantages and overall utility of the method in elaborating drug structure activity relationships are exemplified in a controlled study with real and simulated data.
Perdicaro DJ, etal., Food Funct. 2017 Oct 18;8(10):3501-3509. doi: 10.1039/c7fo01062a.
Metabolic syndrome (MetS) is a risk factor for sudden cardiac death in humans, but animal models are needed for the study of this association. Grape pomace (GP), obtained from the winemaking process, contains phenolic compounds with potential cardioprotective ef
fects. The aim of this study was to evaluate if a high-fat-fructose (HFF) diet facilitates the occurrence of arrhythmias during the reperfusion, and if a GP supplementation could counteract these effects. Wistar rats were fed with control (Ctrl), HFF diet and HFF plus GP (1 g kg-1 day-1) for six weeks. The HFF diet induces characteristic features of MetS (higher systolic blood pressure, dyslipidemia and insulin resistance) which was attenuated by GP supplementation. In addition, HFF induced increased reperfusion arrhythmias that were reduced upon GP supplementation. GP also reduced the non-phosphorylated form of connexin-43 (Cx43) while enhancing heart p-AKT and p-eNOS protein levels and reducing Nox4 levels enhanced by the HFF diet, indicating that GP may increase NO bioavailability in the heart. We found a murine model of MetS with increased arrhythmogenesis and translational value. Furthermore, GP prevents diet-induced heart dysfunction and metabolic alterations. These results highlight the potential utilization of winemaking by-products containing significant amounts of bioactive compounds to prevent/attenuate MetS-associated cardiovascular pathologies.
Raina K, etal., Cancer Res. 2007 Jun 15;67(12):5976-82.
Prostate cancer chemoprevention is an alternative and potential strategy to control this malignancy. Herein, we evaluated the chemopreventive efficacy of grape seed extract (GSE) against prostate cancer in transgenic adenocarcinoma of the mouse prostate (TRAMP)
mice where animals were fed with GSE by oral gavage at 200 mg/kg body weight dose during 4 to 28 weeks of age. Our results showed a significant reduction (46%, P < 0.01) in the weight of genitourinary tract organs in the GSE-fed mice. The GSE-fed group of mice had a higher incidence of prostatic intraepithelial neoplasia but showed strong reduction in the incidence of adenocarcinoma compared with mice in control group. Prostate tissue from the GSE group showed approximately 50% (P < 0.001) decrease in proliferating cell nuclear antigen (PCNA)-positive cells and 64% (P < 0.01) reduction in total PCNA protein level compared with the control group; however, GSE increased apoptotic cells by 8-fold. Furthermore, GSE strongly decreased the protein levels of cyclin B1, cyclin A, and cyclin E by 84% (P < 0.05), 96% (P < 0.05), and 89% (P < 0.001), respectively. The protein expression of cyclin-dependent kinases 2 and 6 and Cdc2 was also decreased by more than 90% (P < 0.05) in the prostate from the GSE-fed group. Together, for the first time, we identified that oral GSE inhibits prostate cancer growth and progression in TRAMP mice, which could be mediated via a strong suppression of cell cycle progression and cell proliferation and an increase in apoptosis.
A low level of high density lipoprotein (HDL) cholesterol is a strong predictor of ischaemic heart disease (IHD) and myocardial infarction. One cause of low HDL-cholesterol is Tangier disease (TD), an autosomal codominant inherited condition first described in 1961 in two siblings on Tangier Island
in the United States of America. Apart from low HDL-cholesterol levels and an increased incidence of atherosclerosis, TD is characterized by reduced total cholesterol, raised triglycerides, peripheral neuropathy and accumulation of cholesteryl esters in macrophages, which causes enlargement of the liver, spleen and tonsils. In contrast to two other monogenic HDL deficiencies in which defects in the plasma proteins apoA-I and LCAT interfere primarily with the formation of HDL (refs 7-10), TD shows a defect in cell signalling and the mobilization of cellular lipids. The genetic defect in TD is unknown, and identification of the Tangier gene will contribute to the understanding of this intracellular pathway and of HDL metabolism and its link with IHD. We report here the localization of the genetic defect in TD to chromosome 9q31, using a genome-wide graphical linkage exclusion strategy in one pedigree, complemented by classical lod score calculations at this region in a total of three pedigrees (combined lod 10.05 at D9S1784). We also provide evidence that TD may be due to a loss-of-function defect.
Yang Z, etal., Nanoscale. 2015 Nov 28;7(44):18725-37. doi: 10.1039/c5nr01172h. Epub 2015 Oct 27.
Current therapies for Alzheimer's disease (AD) can provide a moderate symptomatic reduction or delay progression at various stages of the disease, but such treatments ultimately do not arrest the advancement of AD. As such, novel approaches for AD treatment and prevention are urgently needed. We her
e provide both experimental and computational evidence that pristine graphene and graphene-oxide nanosheets can inhibit Abeta peptide monomer fibrillation and clear mature amyloid fibrils, thus impacting the central molecular superstructures correlated with AD pathogenesis. Our molecular dynamics simulations for the first time reveal that graphene nanosheets can penetrate and extract a large number of peptides from pre-formed amyloid fibrils; these effects seem to be related to exceptionally strong dispersion interactions between peptides and graphene that are further enhanced by strong pi-pi stacking between the aromatic residues of extracted Abeta peptides and the graphene surface. Atomic force microscopy images confirm these predictions by demonstrating that mature amyloid fibrils can be cut into pieces and cleared by graphene oxides. Thioflavin fluorescence assays further illustrate the detailed dynamic processes by which graphene induces inhibition of monomer aggregation and clearance of mature amyloid fibrils, respectively. Cell viability and ROS assays indicate that graphene oxide can indeed mitigate cytotoxicity of Abeta peptide amyloids. Our findings provide new insights into the underlying molecular mechanisms that define graphene-amyloid interaction and suggest that further research on nanotherapies for Alzheimer's and other protein aggregation-related diseases is warranted.
Zhou Y, etal., Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Feb;32(2):173-6.
OBJECTIVE: To explore the effect of grape seed proanthocyanidins (GSPs) on the invasion and migration of A549 lung cancer cells and the underlying mechanism. METHODS: Trypan blue dye exclusion assay was used to determine the cytotoxic effect
of varying doses of GSPs on the BEAS-2B normal human pulmonary epithelial cells. After treated with 0, 10, 20, 40, 80 µg/mL GSP, the proliferation of A549 cells was detected by MTT assay; the invasion and migration of A549 cells were determined by Transwell(TM) assay and scratch wound assay, respectively. The levels of epithelial growth factor receptor (EGFR), E-cadherin, N-cadherin in A549 cells treated with GSPs were detected by Western blotting. RESULTS: (0-40) µg/mL GSPs had no significant toxic effect on BEAS-2B cells, while 80 µg/mL GSPs had significant cytotoxicity to BEAS-2B cells. The proliferation of A549 cells was significantly inhibited within limited dosage in a dose-dependent manner, and the abilities of invasion and migration of A549 cells were also inhibited. Western blotting showed that the expression of EGFR and N-cadherin decreased, while E-cadherin increased after GSPs treatment. CONCLUSION: GSPs could inhibit the abilities of proliferation, invasion and migration of A549 cells, which might be related to the dow-regulation of EGFR and N-cadherin and the up-regulation of E-cadherin.
Zhang Y, etal., J Vasc Surg. 2011 Mar;53(3):743-53. doi: 10.1016/j.jvs.2010.09.017. Epub 2010 Nov 20.
OBJECTIVE: Proanthocyanidins are abundantly found in grape seeds and have been suggested to inhibit the pathogenesis of systemic diseases. We investigated the antithrombotic effects of proanthocyanidins in a rat model of deep vein thrombosis (DVT) and examined t
he underlying mechanisms. METHODS: DVT was induced in rat model by inferior vena cava (IVC) ligation. Grape seed proanthocyanidins extract (GSPE, 400 mg/kg/d) dissolved in saline (2 mL) was orally administered to the experimental rats. Control rats were administrated saline (2 mL) only. The thrombi were harvested and weighed. The IVC was analyzed histologically and by transmission electron microscopy. The cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha) were detected by enzyme-linked immunosorbent assay. Expression of cellular adhesion molecules (CAMs) in thrombi was examined by Western blot. RESULTS: GSPE significantly reduced thrombus length and weight (P < .01) and protected the integrity of the endothelium. GSPE inhibited thrombogenesis-promoting factors P-selectin, von Willebrand factor, and CAMs, and promoted thrombogenesis-demoting factors CD34, vascular endothelial growth factor receptor-2, and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type one motif, member 13). Compared with the control, GSPE significantly lowered the cytokines IL-6 (74.19 +/- 13.86 vs 189.54 +/- 43.76 pg/mL; P < .01), IL-8 (80.71 +/- 21.42 vs 164.56 +/- 39.54 pg/mL; P < .01), and TNF-alpha (43.11 +/- 17.58 vs 231.84 +/- 84.11 pg/mL; P < .01). CONCLUSIONS: GSPE significantly inhibited the propagation of thrombus induced by IVC ligation in a rat model. The antithrombotic properties of proanthocyanidins are likely to be directly associated with endothelial protection and regeneration, platelet aggregation, and inhibition of inflammatory cell and thrombus adhesion. Thus, proanthocyanidins may have a clinical application in DVT treatment.
Almomen SM, etal., Nutrients. 2017 Mar 31;9(4):345. doi: 10.3390/nu9040345.
Individuals living with metabolic syndrome (MetS) such as diabetes and obesity are at high risk for developing chronic kidney disease (CKD). This study investigated the beneficial effect of whole grape powder (WGP) diet on MetS-associated CKD. Obese diabetic ZSF
1 rats, a kidney disease model with MetS, were fed WGP (5%, w/w) diet for six months. Kidney disease was determined using blood and urine chemical analyses, and histology. When compared to Vehicle controls, WGP intake did not change the rat bodyweight, but lowered their kidney, liver and spleen weight, which were in parallel with the lower serum glucose and the higher albumin or albumin/globin ratio. More importantly, WGP intake improved the renal function as urination and proteinuria decreased, or it prevented kidney tissue damage in these diabetic rats. The renal protection of WGP diet was associated with up-regulation of antioxidants (Dhcr24, Gstk1, Prdx2, Sod2, Gpx1 and Gpx4) and downregulation of Txnip (for ROS production) in the kidneys. Furthermore, addition of grape extract reduced H₂O₂-induced cell death of cultured podocytes. In conclusion, daily intake of WGP reduces the progression of kidney disease in obese diabetic rats, suggesting a protective function of antioxidant-rich grape diet against CKD in the setting of MetS.
Durak I, etal., Life Sci. 2005 May 6;76(25):2995-3000. doi: 10.1016/j.lfs.2004.11.018. Epub 2005 Jan 28.
Effects of extract of dried whole black grape including seed on adenosine deaminase (ADA), 5' nucleotidase (5'NT) and xanthine oxidase (XO) enzymes were investigated in cancerous and non-cancerous human colon tissues. Enzyme activities were measured in 20 colon
tissues, 10 from cancerous region and 10 from non cancerous region with and without pre incubation with black grape extract. ADA and 5'NT activities were found increased and that of the XO decreased in the cancerous tissues relative to non cancerous ones. After incubation period with black grape extract for 12 h, ADA and 5'NT activities were found to be significantly lowered but that of XO unchanged in both cancerous and non cancerous tissues. Results suggest that ADA and 5'NT activities increase but XO activity decreases in cancerous human colon tissues, which may provide advantage to the cancerous tissues in obtaining new nucleotides for rapid DNA synthesis through accelerated salvage pathway activity. Black grape extract makes significant inhibition on the ADA and 5'NT activities of cancerous and non cancerous colon tissues, thereby eliminating this advantage of cancer cells, which might be the basis for the beneficial effect of black grape in some kinds of human cancers.
The present study was done to evaluate the role of grape seed proanthocyanidins (GSPs) in isoproterenol (ISO)-induced myocardial injury in rats. Male albino Wistar rats were pretreated with GSP (50, 100, and 150 mg/kg), 6 days a week, for 5 weeks. Induction of r
ats with ISO (85 mg/kg body weight, intraperitoneally) for 2 days resulted in a significant elevation of thiobarbituric acid-reactive substances in serum, mitochondrial cholesterol, triglycerides, and free fatty acids. A significant decrease was observed in serum reduced glutathione; ascorbic acid; alpha-tocopherol; ceruloplasmin; and mitochondrial cytochromes (b, c, c1, and aa3), phospholipids, and adenosine triphosphate. Pretreatment with GSP (100 and 150 mg/kg) positively altered the levels of all the parameters studied and restored normal mitochondrial function when compared with ISO-induced rats. The effect at a dose of 50 mg/kg was not promising when compared with the other 2 doses (100 and 150 mg/kg). These results confirm the efficacy of GSP in alleviating ISO-induced myocardial injury.
Sfaxi I, etal., Can J Physiol Pharmacol. 2015 Jul 29:1-9.
Arsenic is a metalloid found in water, soil, and air from natural and anthropogenic sources, and is commonly found in inorganic as well as organic forms. The clinical use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL) is limited by its cardiotoxic side effects. ... (more)
pan style='font-weight:700;'>Grape seed and skin extract (GSSE) is a polyphenolic mixture with antioxidant properties. This study aimed to evaluate the protective effect of GSSE on arsenic-induced cardiac oxidative stress and injury. Animals exposed to 2.5 mg/kg As2O3 for 21 days exhibited a relevant increase in heart lipoperoxidation, protein carbonylation, and inflammation, as well as a drop in the activity of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). In addition, As2O3 disturbed heart lipidemia and lipase activity, transition metals distribution and the associated enzymes, intracellular mediators such as calcium and the associated calpain activity, as well as myocardial architecture. Treatment with 4 g/kg GSSE protected against most of the deleterious effects provoked by As2O3. Our data suggest that GSSE has the potential to protect against As2O3-induced cardiotoxicity.
Grape polyphenols are known to protect neurons against oxidative stress. We used grape seed extract (GSE) from "Koshu" grapes (Vitis vinifera) containing a variety of polyphenols, and pe
rformed transcriptome analysis to determine the effects of GSE on primary cultures of astrocytes in the hippocampus. GSE upregulated various mRNAs for cytokines, among which interleukin-6 (IL-6) showed the biggest increase after treatment with GSE. The GSE-evoked increase in IL-6 mRNAs was confirmed by quantitative RT-PCR. We also detected IL-6 proteins by ELISA in the supernatant of GSE-treated astrocytes. We made an oxidative stress-induced neuronal cell death model in vitro using a neuron rich culture of the hippocampus. Treatment of the neurons with H(2)O(2) caused neuronal cell death in a time- and concentration-dependent manner. Exogenously applied IL-6 protected against the H(2)O(2)-induced neuronal cell death, which was mimicked by endogenous IL-6 produced by GSE-treated astrocytes. Taken together, GSE acting on astrocytes increased IL-6 production, which functions as a neuroprotective paracrine, could protect neuronal cells from death by oxidative stress.
OBJECTIVE: To study possible inhibition of oxidative stress and cataract formation by single combined formulation of grape seed extract and Zincovit tablets against sodium selenite-induced age-related cataract in Wistar rat pups. METHODS: Oxidative stress and c
onsequent cataract formation was induced by subcutaneous administration of a single dose of sodium selenite (10 micromoles/kg) to Wistar rat pups on day 7 post-natally. In experiments designed to inhibit such cataract formation, the pups were pretreated subcutaneously with combined formulation of grape seed extract and Zincovit tablets (40, 80 and 160 mg/kg), one day prior to the administration of selenite and continuing such treatment till day 20, when the experiments were terminated. The extent of tissue damage caused by the selenite was assessed biochemically by measurements of the levels of reduced glutathione, glutathione peroxidase, glucose-6-phosphate dehydrogenase, protein thiol, catalase, superoxide dismutase, malondialdehyde, aldose reductase, sorbitol dehydrogenase and adenosine triphosphate in the isolated lenses. Cataract formation and its prevention were monitored by examining the eye with pen light illumination and subsequent photography of the isolated lenses. RESULTS: Injection of selenite led to a significant loss of lens clarity due to cataract formation. In the group treated with combined formulation of grape seed extract and Zincovit tablets, the formation of cataract was significantly prevented. In the normal and selenite induced senile cataract control group, the levels of lens oxidative stress markers, G6PD and ATP were substantially lower than in the grape seed extract with Zincovit tablets treated group (p < 0.05). CONCLUSION: Over all, the results suggest that single combined formulation of grape seed extract and Zincovit tablets may offer a prophylactic measure against onset and progression of age- related cataract of human subjects as nutritional food supplement.
Li X, etal., Nephron Exp Nephrol. 2009;111(2):e31-41. Epub 2009 Jan 13.
BACKGROUND: Diabetic nephropathy (DN) is the most common cause of end-stage renal failure. Grape seed proanthocyanidin extracts (GSPE) are powerful antioxidants. However, the role of GSPE on advanced glycation end products (AGEs), receptor for advanced glycation
end products (RAGE) and expression of connective tissue growth factor (CTGF) in DN has not been elucidated. Using streptozotocin-induced diabetic rats, we evaluated the effects of GSPE in DN. METHODS: Wistar rats were induced into diabetes using streptozotocin injections and diabetic rats were treated with GSPE (dosage: 500 mg x kg(-1) x day(-1)) for 24 weeks. The renal pathological changes were examined with PAS staining and electron microscope. The mRNA and protein expression of RAGE and CTGF in kidney were detected by RT-PCR, Western blot and immunohistochemical staining. RESULTS: Treated animals showed reduction in serum AGEs (p < 0.01), proteinuria (p < 0.01) and systolic blood pressure (p < 0.01). GSPE reduced the expression of RAGE (p < 0.01) and CTGF (p < 0.01) in the kidney, which were contributing to reversal of extracellular matrix accumulation in DN. CONCLUSION: Our results suggest that GSPE hold substantial promise for the treatment of DN. GSPE can decrease proteinuria, attenuating the progression of nephropathy in diabetic rats. Renoprotective effects of GSPE are correlated with suppression on AGEs/RAGE axis, downregulating expression of CTGF.
Hahn-Obercyger M, etal., J Agric Food Chem. 2005 Mar 9;53(5):1828-32.
Citrus fruits are considered to be functional foods that promote good health. This study was carried out to assess the effect of oroblanco and grapefruit consumption on hepatic detoxification enzymes. Male Sprague-Dawley rats were provided with either regular dr
inking water (control) or experimental treatments of oroblanco juice, grapefruit juice, or a sugar mix for 6 weeks. After 1 week of treatment, half the animals in each group were injected with the procarcinogen 1,2-dimethylhydrazine. Grapefruit juice significantly increased activity and expression of the hepatic phase I enzyme, cytochrome P450 CYP1A1, with a marked trend toward enhanced NAD(P)H:quinone reductase (QR) activity. Oroblanco juice significantly increased glutathione S-transferase phase II enzyme activity along with CYP1A1 expression and a notable trend toward increased activity of both CYP1A1 and QR. These results suggest that these citrus fruits are bifunctional inducers, modulating both phase I and phase II drug-metabolizing enzymes to enhance hepatic detoxification.
The present work investigates the modulation of grapefruit flavonoid naringenin over liver X receptor alpha (LXRα) and its target genes in THP-1 macrophages, focusing on AMP-activated protein kinase (AMPK) implication. Naringenin induced LXRα at mRNA
and protein levels besides influencing the expression of LXRα target genes ABCA1, ABCG1 (ATP-binding cassette A1 and G1), and SREBP1c (sterol response element binding protein 1c) in THP-1 macrophages. The increased LXRα mRNA and protein expression was reverted when AMPK was inhibited by its chemical inhibitor, compound C or by transfection with AMPK α1 and α2 siRNA. Naringenin treatments were also able to promote reverse cholesterol transport in THP-1 cells, which is in line with the increase in the ABCA1 and ABCG1 expression found. Treatments with this flavonoid also inhibited cell migration in THP-1 cells. In conclusion, LXRα and its target genes are up-regulated by naringenin in an AMPK dependent manner in human macrophages. The enhancement in the expression of genes involved in cholesterol efflux may reveal a new mechanism by which this polyphenol can prevent atherosclerosis and foam cell progression.
Ismail AF, etal., J Photochem Photobiol B. 2016 Apr 4;160:1-10. doi: 10.1016/j.jphotobiol.2016.03.027.
Carbon tetrachloride (CCl4) and ionizing radiation are well known environmental pollutants that generate free radicals and induce oxidative stress. The liver is the primary and major target organ responsible for the metabolism of drugs, toxic chemicals and affected by irradiation. This study invest
igated the effect of grape seed oil (GSO) on acute liver injury induced by carbon tetrachloride (CCl4) in gamma-irradiated rats (7Gy). CCl4-intoxicated rats exhibited an elevation of ALT, AST activities, IL-6 and TNF-alpha level in the serum. Further, the levels of MDA, NO, NF-kappaB and the gene expression of CYP2E1, iNOS and Caspase-3 were increased, and SOD, CAT, GSH-Px, GST activities and GSH content were decreased. Furthermore, silent information regulator protein 1 (SIRT1) gene expression was markedly down-regulated. Additionally, alterations of the trace elements; copper, manganese, zinc and DNA fragmentation was observed in the hepatic tissues of the intoxicated group. These effects were augmented in CCl4-intoxicated-gamma-irradiated rats. However, the administration of GSO ameliorated these parameters. GSO exhibit protective effects on CCl4 induced acute liver injury in gamma-irradiated rats that could be attributed to its potent antioxidant, anti-inflammatory and anti-apoptotic activities. The induction of the antioxidant enzymes activities, down-regulation of the CYP2E1, iNOS, Caspase-3 and NF-kappaB expression, up-regulation of the trace elements concentration levels and activation of SIRT1 gene expression are responsible for the improvement of the antioxidant and anti-inflammatory status in the hepatic tissues and could be claimed to be the hepatoprotective mechanism of GSO.
Walter A, etal., FASEB J. 2010 Sep;24(9):3360-9. doi: 10.1096/fj.09-149419. Epub 2010 May 4.
This study evaluated the in vivo antitumor activity of grape-derived polyphenols. BALB/c mice were subcutaneously implanted with C26 colon carcinoma cells, and 2 d later they received either solvent or red wine polyphenols (RWPs) (100 mg/kg/d, human equivalent d
ose approximately 500 mg/d) in the drinking water for 25 d. Wistar rats received either solvent or RWPs (100 mg/kg/d, human equivalent dose approximately 1000 mg/d) in the drinking water 1 wk before injection of azoxymethane and were studied 10 wk later. In mice, RWPs inhibited tumor growth by 31%, reduced tumor vascularization and the number of lung metastases, decreased proliferation as indicated by down-regulation of Ki67, cyclin D1, and UHRF1, and increased apoptosis as indicated by TUNEL staining and active caspase-3 levels in tumor cells. RWPs reduced expression of VEGF, matrix metalloproteinase (MMP)-2, MMP-9, and cyclooxygenase-2 and increased expression of tumor suppressor genes p16(INK4A), p53, and p73 in tumor cells. In rats, RWPs reduced by 49% the number of azoxymethane-induced aberrant crypt foci (preneoplastic lesions) in colon. Thus, RWPs effectively reduced the development of colon carcinoma tumors in vivo by blunting tumor vascularization and by inhibiting proliferation and promoting apoptosis of tumor cells subsequent to an up-regulation of tumor suppressor genes.
Songtawee N, etal., J Mol Graph Model. 2015 May;58:16-29. doi: 10.1016/j.jmgm.2015.03.002. Epub 2015 Mar 14.
Abnormal activation of EGFR is associated with human cancer, and thus it is a key target for inhibition in cancer therapy. There is evidence suggesting that the activation mechanism of EGFR is based upon the formation of the asymmetric dimer of the kinase domains. Here, we performed MD simulations
on the asymmetric dimer for both active and inactive conformations of EGFR kinase domain to investigate flexibility and intrinsic motions of the proteins. Simulations of the active conformation showed that the formation of the asymmetric dimer changes the dynamics of EGFR kinase domain by suppressing fluctuation of the protein and altering the direction of motion of the protein. In contrast, the asymmetric dimerization of the inactive conformation does not alter the overall fluctuation of the kinase domain and does not initiate destabilizing of the inactive structure. We also investigated the intermolecular interactions in the EGFR asymmetric dimers and found that in the active conformation the interactions are dominated by loop-loop contacts rather than those from the helix-helix interactions. In contrast, helix-helix interaction seemed to be more significant for the inactive kinase structure. This work helps us to better understand the conformational flexibility and dynamics of the EGFR kinase domain, as well as provides information that may be useful to develop newer classes of inhibitors that can block allosteric sites rather than the more traditional catalytic site.
Shi Y, etal., ACS Appl Mater Interfaces. 2015 May 27;7(20):10935-43. doi: 10.1021/acsami.5b02199. Epub 2015 May 12.
Circulating tumor cells (CTCs) are extremely rare cells in blood containing billions of other cells. The selective capture and identification of rare cells with sufficient sensitivity is a real challenge. Driven by this need, this manuscript reports the development of a multifunctional biocompatible
graphene oxide quantum dots (GOQDs) coated, high-luminescence magnetic nanoplatform for the selective separation and diagnosis of Glypican-3 (GPC3)-expressed Hep G2 liver cancer tumor CTCs from infected blood. Experimental data show that an anti-GPC3-antibody-attached multifunctional nanoplatform can be used for selective Hep G2 hepatocellular carcinoma tumor cell separation from infected blood containing 10 tumor cells/mL of blood in a 15 mL sample. Reported data indicate that, because of an extremely high two-photon absorption cross section (40530 GM), an anti-GPC3-antibody-attached GOQDs-coated magnetic nanoplatform can be used as a two-photon luminescence platform for selective and very bright imaging of a Hep G2 tumor cell in a biological transparency window using 960 nm light. Experimental results with nontargeted GPC3(-) and SK-BR-3 breast cancer cells show that multifunctional-nanoplatform-based cell separation, followed by two-photon imaging, is highly selective for Hep G2 hepatocellular carcinoma tumor cells.
We report results of the studies relating to fabrication of a non-invasive, label-free and an efficient biosensing platform for detection of the oral cancer biomarker (CYFRA-21-1). One step hydrothermal process was used for uniform decoration of nanostructured zirconia (average particle size 13 nm)
on reduced graphene oxide (ZrO2-RGO) to avoid coagulation of the zirconia nanoparticles and to obtain enhanced electrochemical performance of ZrO2-RGO nanocomposite based biosensor. Further, ZrO2-RGO has been functionalized using 3-aminopropyl triethoxy saline (APTES) and electrophoretically deposited on the indium tin oxide coated glass substrate at a low DC potential.The APTES/ZrO2-RGO/ITO electrode exhibits improved heterogeneous electron transfer (more than two times) with respect to that of the APTES/ZrO2/ITO electrode indicating faster electron transfer kinetics. The -NH2 containing APTES/ZrO2-RGO/ITO platform is further biofunctionalized with anti-CYFRA-21-1. The structural and morphological investigations of the ZrO2-RGO based biosensing platform have been accomplished using X-ray diffraction (XRD), electrochemical, transmission electron microscopy (TEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FT-IR) studies. This immunosensor exhibits a wider linear detection range (2-22 ng mL(-1)), excellent sensitivity (0.756 microA mL ng(-1)) and a remarkable lower detection limit of 0.122 ng mL(-1). The observed results have been validated via enzyme linked immunosorbent assay (ELISA).
Many cancer chemotherapy agents act by targeting the DNA of cancer cells, causing substantial damage within their genome and causing them to undergo apoptosis. An effective DNA repair pathway in cancer cells can act in a reverse way by removing these drug-induced DNA lesions, allowing cancer cells
to survive, grow and proliferate. In this context, DNA repair inhibitors opened a new avenue in cancer treatment, by blocking the DNA repair mechanisms from removing the chemotherapy-mediated DNA damage. In particular, the nucleotide excision repair (NER) involves more than thirty protein-protein interactions and removes DNA adducts caused by platinum-based chemotherapy. The excision repair cross-complementation group 1 (ERCC1)-xeroderma pigmentosum, complementation group A (XPA) protein (XPA-ERCC1) complex seems to be one of the most promising targets in this pathway. ERCC1 is over expressed in cancer cells and the only known cellular function so far for XPA is to recruit ERCC1 to the damaged point. Here, we build upon our recent advances in identifying inhibitors for this interaction and continue our efforts to rationally design more effective and potent regulators for the NER pathway. We employed in silico drug design techniques to: (1) identify compounds similar to the recently discovered inhibitors, but more effective at inhibiting the XPA-ERCC1 interactions, and (2) identify different scaffolds to develop novel lead compounds. Two known inhibitor structures have been used as starting points for two ligand/structure-hybrid virtual screening approaches. The findings described here form a milestone in discovering novel inhibitors for the NER pathway aiming at improving the efficacy of current platinum-based therapy, by modulating the XPA-ERCC1 interaction.
Wang YH, etal., Int Immunopharmacol. 2011 Oct;11(10):1620-7. doi: 10.1016/j.intimp.2011.05.024. Epub 2011 Jun 12.
The aim of this study was to elucidate the molecular mechanisms involved in the therapeutic effects of proanthocyanidins from grape seeds (GSPE) on recurrent ulcerative colitis (UC) in rats. GSPE in doses of 100, 200, and 400mg/kg were intragastrically administe
red per day for 7 days after recurrent colitis was twice-induced by TNBS. The levels of GSH, as well as the activity of GSH-Px and SOD in colon tissues were measured by biochemical methods. The expression levels of tumor necrosis factor-alpha (TNF-alpha) and the nuclear translocation levels of nuclear factor-kappa B (NF-kappaB) in the colon tissues were measured by enzyme-linked immunosorbent assay methods. Western blotting analysis was used to determine the protein expression levels of inhibitory kappa B-alpha (IkappaBalpha), inhibitor kappa B kinase (IKKalpha/beta), phosphorylated IkappaBalpha and phosphorylated IKKalpha/beta. GSPE treatment was associated with a remarkable increased the activity of GSH-Px and SOD with GSH levels in TNBS-induced recurrent colitis rats as compared to the model group. GSPE also significantly reduced the expression levels of TNF-alpha, p-IKKalpha/beta, p-IkappaBalpha and the translocation of NF-kappaB in the colon mucosa. GSPE exerted a protective effect on recurrent colitis in rats by modifying the inflammatory response and promoting damaged tissue repair to improve colonic oxidative stress. Moreover, GSPE inhibited the TNBS-induced inflammatory of recurrent colitis though blocking NF-kappaB signaling pathways.
Safwen K, etal., Int J Stroke. 2015 Apr;10(3):415-24. doi: 10.1111/ijs.12391. Epub 2014 Nov 3.
Ischemic stroke is a leading cause of long lasting disability in humans and oxidative stress an important underlying cause. The present study aims to determine the effect of short term (seven-days) administration of high dosage grape seed and skin extract (GSSE
2.5 g/kg) on ischemia/reperfusion (I/R) injury in a rat model of global ischemia. Ischemia was induced by occlusion of the common carotid arteries for 30 min followed by one-hour reperfusion on control or GSSE treated animals. I/R induced a drastic oxidative stress characterized by high lipid and protein oxidation, a drop in antioxidant enzyme defenses, disturbed transition metals as free iron overload and depletion of copper, zinc and manganese as well as of associated brain enzyme activities as glutamine synthetase and lactate dehydrogenase. I/R also induced NO and calcium disruption and an increase in calpain activity, a calcium-sensitive cysteine protease. Interestingly, almost all I/R-induced disturbances were prevented by GSSE pretreatment as oxidative stress, transition metals associated enzyme activities, brain damage size and histology. Owing to its antioxidant potential, high dosage GSSE protected efficiently the brain against ischemic stroke and should be translated to humans.
Cisplatin is one of the most potent chemotherapeutic antitumor drugs. Oxidative stress has been proven to be involved in cisplatin-induced toxicity. Therefore, the present study was undertaken to examine the antioxidant potential of grape seed proanthocyanidin e
xtract (GSPE) against the toxicity of cisplatin in male rats. Cisplatin treated animals revealed a significant elevation in plasma, heart, kidney and liver thiobarbituric acid reactive substances (TBARS), while the activities of antioxidant enzymes (GST, SOD, CAT and GSH-Px, and the levels of glutathione (GSH) were decreased. Aspartate and alanine transaminases (AST and ALT), creatine kinase and lactate dehydrogenase were significantly increased in plasma, while liver AST and ALT were significantly decreased. Cisplatin significantly increased the levels of plasma total lipid, cholesterol, urea and creatinine, and the relative weight of kidney. On the other hand, plasma total protein and albumin, and body weight were significantly decreased. GSPE reduced cisplatin-induced the levels of TBARS in plasma, heart, kidney and liver, TL, cholesterol, urea and creatinine, and liver AST and ALT. Moreover, it ameliorated cisplatin-induced decrease in the activities of antioxidant enzymes, and GSH, total protein and albumin. Therefore, the present results revealed that GSPE exerts a protective effect by antagonizing cisplatin toxicity.
Lu M, etal., J Nutr Sci Vitaminol (Tokyo). 2010;56(2):87-97.
Diabetic encephalopathy is a severe complication in patients with long-term hyperglycemia. Oxidative stress is thought to be closely implicated in this disorder, so in this study, we examined whether grape seed proanthocyanidin extract (GSPE), a naturally occurr
ing antioxidant derived from grape seeds, could reduce the injuries in the cerebral cortex of diabetic rats by modulating advanced glycation end products (AGEs)/the receptor for AGEs (RAGE)/nuclear factor-kappa B p65 (NF-kappaB p65) pathway, which is crucial in oxidative stress. Body weight and serum AGEs were tested; cerebral cortexes were isolated for morphological observations and the pyramidal cell layers were immunohistochemically stained for the detection of RAGE, NF-kappaB p65, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) as well. For RAGE and NF-kappaB p65, quantitative reverse transcriptase coupled to polymerase chain reaction (RT-PCR) was employed for determination of mRNA levels, and western blot was used to detect protein expression. Our results showed that long term hyperglycemia in diabetic rats caused the degeneration of neurons and the up-regulation of serum AGEs, and also the up-regulation of RAGE, NF-kappaB p65, VCAM-1 and ICAM-1 in the brain. We found that GSPE treatment improved the pathological changes of diabetic rats by modulating the AGEs/RAGE/NF-kappaB p65 pathway. This study enables us to further understand the key role that the AGEs/RAGE/NF-kappaB pathway plays in the pathogenesis of diabetic encephalopathy, and confirms that GSPE might be a therapeutical means to the prevention and treatment of this disorder.
Several examples of allosteric modulators of GPCRs have been reported recently in the literature, but understanding their molecular mechanism presents a new challenge for medicinal chemistry. For the specific case of the cellular receptor CXCR4, it is known that pepducins (lipidated fragments of int
racellular GPCR loops) such as ATI-2341 modulate CXCR4 activity agonistically via an allosteric mechanism. Moreover, there are also examples of small organic molecules such as AMD11070 and GSK812397 which may also act as allosteric antagonists. However, incomplete knowledge of the ligand-binding sites has hampered a detailed molecular understanding of how these inhibitors work. Here, we attempt to answer this question by analysing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose two different allosteric binding sites, one located in the intracellular loops 1, 2 and 3 (ICL1, ICL2 and ICL3) which binds the pepducin agonist ATI-2341, and the other at a subsite of the main extracellular orthosteric binding pocket between extracellular loops 1 and 2 and the N-terminus, which binds the antagonists AMD11070 and GSK812397. Allosteric interactions between the CXCR4 and ATI-2341 were predicted by combining different modeling approaches. First, a rotational blind docking search was applied and the best poses were subsequently refined using flexible docking methods and molecular dynamic simulations. For the AMD11070 and GSK812397 antagonists, the entire CXCR4 protein surface was explored by blind docking in order to define the binding region. A second docking analysis by subsites was then performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 allosteric modulators.
Prolonged hypertension is the leading cause of heart failure. Failing hearts show reduced peroxisome proliferator-activating receptor (PPAR) activity and enhanced nuclear factor kappaB (NF-kappaB) activity, which together modify cardiac inflammation and fibrosis. In vitro studies suggest that phyto
chemicals alter PPAR and NF-kappaB activity, but the capabilities of a phytochemical-rich diet are less understood. Grapes contain an array of commonly consumed dietary phytochemicals. In Dahl salt-sensitive hypertensive rats, we showed previously that dietary provision of whole table grape powder (3% weight:weight) for 18 weeks reduced blood pressure, cardiac hypertrophy, and diastolic dysfunction. The hypothesis tested here is that, in this model, phytochemical provision from whole grape powder impacts cardiac PPAR and NF-kappaB activity and their related gene transcripts. Grape-fed rats had enhanced PPAR-alpha and PPAR-gamma DNA binding activity but reduced NF-kappaB DNA binding activity. RT-PCR revealed that grape-fed rats showed upregulated mRNA for PPAR-alpha, PPAR-gamma coactivator-1alpha, PPAR-gamma, and the cytosolic NF-kappaB inhibitor, inhibitor-kappaBalpha. By contrast, grape-fed rats showed downregulated mRNA for tumor necrosis factor-alpha and transforming growth factor-beta1. Finally, grape-fed rats showed significantly reduced cardiac tumor necrosis factor-alpha and transforming growth factor-beta protein expression, increased inhibitor-kappaBalpha expression, and reduced cardiac fibrosis. In the Dahl salt-sensitive rat, chronic intake of grapes altered cardiac transcripts related to PPAR and NF-kappaB that may be significant to the observed diet-associated cardioprotection.
