ABSTRACT
Soil and water samples were collected from various regions of SIPCOT and nearby Vanappadi Lake, Ranipet, Tamilnadu, India. Based on their colony morphology and their stability during subculturing, 72 bacteria were isolated, of which 14 isolates were actinomycetes. Preliminary selection was carried out to exploit the ability of the microorganisms to utilize sodium cyanate as nitrogen source. Those organisms that were able to utilize cyanate were subjected to secondary screening viz., utilization of sodium cyanide as the nitrogen source. The oxygenolytic cleavage of cyanide is dependent on cyanide monooxygenase which obligately requires pterin cofactor for its activity. Based on this, the organisms capable of utilizing sodium cyanide were tested for the presence of pterin. Thin layer chromatography (TLC) of the cell extracts using n-butanol: 5 N glacial acetic acid (4:1) revealed that 10 out of 12 organisms that were able to utilize cyanide had the pterin-related blue fluorescent compound in the cell extract. The cell extracts of these 10 organisms were subjected to high performance thin layer chromatography (HPTLC) for further confirmation using a pterin standard. Based on the incubation period, cell biomass yield, peak height and area, strain VPW3 was selected and was identified as Bacillus subtilis. The Rf value of the cell extract was 0.73 which was consistent with the 0.74 Rf value of the pterin standard when scanned at 254 nm. The compound was extracted and purified by preparative High Performance Liquid Chromatography (HPLC). Characterization of the compound was performed by ultraviolet spectrum, fluorescence spectrum, Electrospray Ionization-Mass Spectrometry (ESI-MS), and Nuclear Magnetic Resonance spectroscopy (NMR). The compound is proposed to be 6-propionyl pterin (2-amino-6-propionyl-3H-pteridin-4-one).
Subject(s)
Bacillus subtilis/chemistry , Bacillus subtilis/metabolism , Cyanides/metabolism , Pterins/chemistry , Bacillus subtilis/isolation & purification , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , India , Magnetic Resonance Spectroscopy , Phylogeny , Soil Microbiology , Spectrometry, Mass, Electrospray Ionization , Water MicrobiologyABSTRACT
Three isostructural metal-organic frameworks, (MOFs), [Fe(OH)(1,4-NDC)] (1), [Al(OH)(1,4-NDC)] (2), and [In(OH)(1,4-NDC)] (3) have been synthesized hydrothermally by using 1,4-naphthalene dicarboxylate (1,4-NDC) as a linker. The MOFs were characterized using various techniques and further used as precursor materials for the synthesis of metal/metal oxide nanoparticles inserted in a carbon matrix through a simple thermal conversion method. The newly synthesized carbon materials were characterized by scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy analysis, powder X-ray diffraction and BET analysis. The results showed that the MOF-derived carbon composite materials maintained the morphology of the original MOF upon carbonization, and confirmed the insertion of metal/metal oxide particles in the carbon matrix.
ABSTRACT
Six new alkaline-earth metal carboxyphosphonates [Mg(H2O)(H2PMIDA)] (1), [Sr(H2O)(H2PMIDA)] (2), [Sr2(H2O)(PMIDA)] (3), [Sr2(HPO4)(H2PMIDA)] (4), [Ba2(HPO4)(H2PMIDA)] (5), and [Ba2(H2O)(H2PMIDA)2] (6) (H4PMIDA = N-(phosphonomethyl)iminodiacetic acid) have been synthesized solvothermally in order to study the coordination behavior of H4PMIDA towards alkaline-earth metal ions (Mg(2+), Sr(2+), and Ba(2+)) and the structural features of the resulting polymeric compounds. The newly synthesized compounds have been characterized by elemental analysis, UV-Vis spectrometry, IR spectroscopy, thermogravimetry analysis, solid state (31)P MAS NMR, powder X-ray diffraction analysis and single crystal X-ray diffraction techniques. The single crystal structure analysis revealed structural variability of the prepared compounds. Compounds 1, 2, 4 and 5 are three-dimensional with the H2PMIDA skeletons connecting the inorganic parts to each other, whereas compound has a layered structure. Compounds 2, 4 and 5 contain helical structural motifs. In addition, the extrinsic luminescent properties of Eu(III)- and Tb(III)-doped compounds 1, 4 and 5 have also been studied.
ABSTRACT
A novel water soluble ligand-bridged cobalt(II) coordination polymer has been synthesized by reacting the new ligand, 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (isonicotinic) hydrazone (H(2)L) with Co(NO(3))(2)·6H(2)O and characterized by spectral, analytical and structural methods. Single crystal X-ray diffraction studies revealed that the Co(II) complex, {[Co(H(2)L)(H(2)O)(2)](NO(3))(2)·3H(2)O}(n) has a slightly distorted octahedral geometry around the central Co(II) ion; the ligand is coordinated through the ONO donor atoms to one Co(II) metal center and bridged through the pyridine nitrogen atom to another similar Co(II) center so as to form a one-dimensional polymeric unit. The interaction of the ligand and the complex with calf thymus DNA (CT-DNA) has been explored by absorption and emission titration methods, which revealed that the compounds could interact with CT-DNA through intercalation. The interactions of the compounds with bovine serum albumin (BSA) were also investigated using UV-visible, fluorescence and synchronous fluorescence spectroscopic methods. The results indicated that the complex exhibited a strong binding to BSA over the ligand. Investigation of the antioxidative properties showed that the polymeric Co(II) complex has a strong radical scavenging potency against hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl radicals, nitric oxide and superoxide anion radicals. Further, the cytotoxic effect of the compounds examined on cancerous cell lines, such as human cervical cancer cells (HeLa), human laryngeal epithelial carcinoma cells (HEp-2), human liver carcinoma cells (Hep G2), human skin cancer cells (A431) and non-cancerous NIH 3T3 mouse embryonic fibroblasts cell lines showed that the complex exhibited substantial anticancer activity.
Subject(s)
Antineoplastic Agents/chemistry , Cobalt/chemistry , Coordination Complexes/chemistry , Free Radical Scavengers/chemistry , Intercalating Agents/chemistry , Animals , Antineoplastic Agents/pharmacology , Cattle , Cell Line , Cell Line, Tumor , Cobalt/pharmacology , Coordination Complexes/pharmacology , Crystallography, X-Ray , DNA/metabolism , Free Radical Scavengers/pharmacology , Humans , Hydrazones/chemistry , Hydrazones/pharmacology , Intercalating Agents/pharmacology , Mice , Models, Molecular , Quinolines/chemistry , Quinolines/pharmacology , Serum Albumin, Bovine/metabolismABSTRACT
Novel 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (4'-methylbenzoyl) hydrazone (H(2)L) (1) and its two copper(II) complexes have been synthesized. Single-crystal X-ray diffraction studies revealed that the structure of the new copper(II) chloride complex, [Cu(H(2)L)Cl(2)]·2H(2)O (2), is square pyramidal and that of the copper(II) nitrate complex, [Cu(HL)NO(3)]·DMF (3), is square planar. In 2, the copper atom is coordinated by the ligand with ONO donor atoms, one chloride ion in the apical position, and the other chloride in the basal plane. In 3, the ligand coordinates as a uninegative tridentate ONO(-) species and with one nitrate ion in the basal plane. DNA binding experiments indicated that the ligand and copper(II) complexes can interact with DNA through intercalation. Bovine serum albumin binding studies revealed that the compounds strongly quench the intrinsic fluorescence of bovine serum albumin through a static quenching process. Antioxidative activity tests showed that 1 and its copper(II) complexes have significant radical scavenging activity against free radicals. Cytotoxic activities of the ligand and copper(II) complexes showed that the two copper(II) complexes exhibited more effective cytotoxic activity against HeLa and HEp-2 cells than the corresponding ligand. The entire biological activity results showed that the activity order was 1 < 2 < 3.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antioxidants/chemistry , Antioxidants/pharmacology , Copper/chemistry , Copper/pharmacology , Hydrazones/chemistry , Hydrazones/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antioxidants/chemical synthesis , Cattle , Cell Line, Tumor , Cell Survival/drug effects , Coordination Complexes/chemical synthesis , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , DNA/metabolism , Humans , Hydrazones/chemical synthesis , Neoplasms/drug therapy , Serum Albumin, Bovine/metabolism , Structure-Activity RelationshipABSTRACT
A new actinomycete strain, isolated from humus soils in the Western Ghats, was found to be an efficient pigment producer. The strain, designated AAA5, was identified as a putative Streptomyces aurantiacus strain based on cultural properties, morphology, carbon source utilization, and analysis of the 16S rRNA gene. The strain produced a reddish-brown pigmented compound during the secondary metabolites phase. A yellow compound was derived from the extracted pigment and was identified as the quinone-related antibiotic resistomycin based on ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, liquid chromatography and mass spectroscopy, and nuclear magnetic resonance analyses. The AAA5 strain was found to produce large quantities of resistomycin (52.5 mg/L). It showed potent cytotoxic activity against cell lines viz. HepG2 (hepatic carcinoma) and HeLa (cervical carcinoma) in vitro, with growth inhibition (GI(50)) of 0.006 and 0.005 µg/ml, respectively. The strain also exhibited broad antimicrobial activities against both Gram-positive and Gram-negative bacteria. Therefore, AAA5 may have great potential as an industrial resistomycin-producing strain.
Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Soil Microbiology , Streptomyces/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/chemistry , Bacteria/drug effects , Benzopyrenes/chemistry , Benzopyrenes/metabolism , Benzopyrenes/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Molecular Sequence Data , Phylogeny , Streptomyces/classification , Streptomyces/genetics , Streptomyces/isolation & purificationABSTRACT
A new ligand, 2-oxo-1,2-dihydroquinoline-3-carbaldehyde semicarbazone (OQsc-H) (1);, its N(4)-phenyl derivative (OQsc-Ph) (2); and their corresponding copper(II) complexes [CuCl(2)(OQsc-H)]·H(2)O·CH(3)OH (3), [CuCl(2)(OQsc-Ph)(H(2)O)]·CH(3)OH (4), and [CuNO(3)(OQsc-Ph)(H(2)O)]NO(3)·H(2)O·C(2)H(5)OH (5) have been synthesized and characterized by structural, analytical, and spectral methods, in order to investigate the influence of N(4)-phenyl substitution on structure and pharmacological properties. In all of the complexes, the ligands coordinated to the Cu(II) ion in a neutral fashion via ONO donor atoms. The single-crystal X-ray structures of neutral complex (3) and cationic complex (5) exhibit a slightly distorted square-pyramidal structure, while neutral complex (4) revealed an octahedral structure. The interaction of the compounds with calf thymus DNA (CT-DNA) has been explored by absorption and emission titration methods, which revealed that compounds 1-5 could interact with CT-DNA through intercalation. A gel electrophoresis pictogram demonstrated the ability of the complexes (3-5) to cleave the pBR322 plasmid DNA through a hydrolytic process. The interactions of the compounds with bovine serum albumin (BSA) were also investigated using UV-visible, fluorescence, and synchronous fluorescence spectroscopic methods. The results indicated that all of the compounds could quench the intrinsic fluorescence of BSA in a static quenching process. Investigations of antioxidative properties showed that all of the compounds have strong radical scavenging potencies against hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl radicals, nitric oxide, and superoxide anion radicals. Further, the cytotoxic effect of the compounds examined on cancerous cell lines such as human cervical cancer cells (HeLa), human laryngeal epithelial carcinoma cells (HEp-2), human liver carcinoma cells (Hep G2), human skin cancer cells (A431), and noncancerous NIH 3T3 mouse embryonic fibroblasts cell lines showed that all three complexes exhibited substantial cytotoxic activity. Further, all of the pharmacological investigations support the fact that there exists a strong influence of N(4)-phenyl substitution in semicarbazone.
Subject(s)
Aldehydes/chemical synthesis , Antineoplastic Agents/chemical synthesis , Antioxidants/chemical synthesis , Coordination Complexes/chemical synthesis , Copper , Semicarbazones/chemical synthesis , Aldehydes/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Cattle , Cell Line, Tumor , Cell Survival/drug effects , Coordination Complexes/pharmacology , Crystallography, X-Ray , DNA/chemistry , DNA Cleavage , HeLa Cells , Humans , Mice , Models, Molecular , Phenols/chemistry , Protein Binding , Semicarbazones/pharmacology , Serum Albumin, Bovine/chemistry , Spectrometry, Fluorescence , Structure-Activity RelationshipABSTRACT
Four 2-oxo-1,2-dihydroquinoline-3-carbaldehyde N-substituted thiosemicarbazone ligands (H(2)-OQtsc-R, where R = H, Me, Et or Ph) and their corresponding new copper(II) complexes [CuCl(2)(H(2)-OQtsc-H)]·2H(2)O (1), [CuCl(2)(H(2)-OQtsc-Me)]·2H(2)O (2), [CuCl(2)(H(2)-OQtsc-Et)(CH(3)OH)]Cl (3) and [CuCl(H-OQtsc-Ph)]·CH(3)OH (4) have been synthesized in order to correlate the effect of terminal N-substitution on coordination behaviour, structure and biological activity. Single crystal X-ray diffraction studies revealed that the complexes 1, 2 and 3 have square pyramidal geometry around the central metal ion. In the complexes 1 and 2, the copper ion is coordinated by the ligand with ONS donor atoms, one chloride ion in apical position and the other chloride in the basal plane. Complex 3 consists of [CuCl(2)(H(2)-OQtsc-Et)(CH(3)OH)](+) cation and a chloride as counter ion. The copper ion is coordinated by the ligand with ONS donor atoms and by one chloride ion in the basal plane. One methanol molecule is bonded through its neutral oxygen in the apical position. Complex 4 is square planar with the ligand coordinating through uni-negative tridentate ONS(-) and by one chloride ion in the basal plane. The binding of complexes with lysozyme protein was carried out by fluorescence spectroscopy. Investigations of antioxidation properties showed that all the copper(II) complexes have strong radical scavenging properties. The cytotoxicity of the complexes 3 and 4 against NIH 3T3 and HeLa cell lines showed that synergy between the metal and ligands results in a significant enhancement in the cell death with IC(50) of ~10-40 µM. A size dependence of substitution at terminal N in the thiosemicarbazones on the biological activities of the complexes has been observed.
Subject(s)
Coordination Complexes/chemistry , Copper/chemistry , Muramidase/chemistry , Thiosemicarbazones/chemistry , Animals , Cell Line , Coordination Complexes/chemical synthesis , Coordination Complexes/toxicity , Crystallography, X-Ray , Humans , Mice , Molecular Conformation , Protein BindingABSTRACT
AIMS: Transforming growth factor-ß (TGF-ß) signaling is critical for the differentiation of smooth muscle cells (SMCs) into quiescent cells expressing a full repertoire of contractile proteins. Heterozygous mutations in TGF-ß receptor type II (TGFBR2) disrupt TGF-ß signaling and lead to genetic conditions that predispose to thoracic aortic aneurysms and dissections (TAADs). The aim of this study is to determine the molecular mechanism by which TGFBR2 mutations cause TAADs. METHODS AND RESULTS: Using aortic SMCs explanted from patients with TGFBR2 mutations, we show decreased expression of SMC contractile proteins compared with controls. Exposure to TGF-ß1 fails to increase expression of contractile genes in mutant SMCs, whereas control cells further increase expression of these genes. Analysis of fixed and frozen aortas from patients with TGFBR2 mutations confirms decreased in vivo expression of contractile proteins relative to unaffected aortas. Fibroblasts explanted from patients with TGFBR2 mutations fail to transform into mature myofibroblasts with TGF-ß1 stimulation as assessed by expression of contractile proteins. CONCLUSIONS: These data support the conclusion that heterozygous TGFBR2 mutations lead to decreased expression of SMC contractile protein in both SMCs and myofibroblasts. The failure of TGFBR2-mutant SMCs to fully express SMC contractile proteins predicts defective contractile function in these cells and aligns with a hypothesis that defective SMC contractile function contributes to the pathogenesis of TAAD.
Subject(s)
Aortic Aneurysm, Thoracic/genetics , Aortic Dissection/genetics , Cell Differentiation/genetics , Genetic Predisposition to Disease/genetics , Muscle, Smooth, Vascular/cytology , Myofibroblasts/cytology , Protein Serine-Threonine Kinases/genetics , Receptors, Transforming Growth Factor beta/genetics , Actins/metabolism , Aortic Dissection/metabolism , Animals , Aortic Aneurysm, Thoracic/metabolism , Calcium-Binding Proteins/metabolism , Calmodulin-Binding Proteins/metabolism , Case-Control Studies , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line , Cell Proliferation , Cells, Cultured , Humans , Mice , Microfilament Proteins/metabolism , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myofibroblasts/metabolism , Phenotype , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/physiology , Transfection , Transforming Growth Factor beta1/pharmacology , CalponinsABSTRACT
OBJECTIVE: This study sought to characterize the inflammatory infiltrate in ascending thoracic aortic aneurysm in patients with Marfan syndrome, familial thoracic aortic aneurysm, or nonfamilial thoracic aortic aneurysm. BACKGROUND: Thoracic aortic aneurysms are associated with a pathologic lesion termed "medial degeneration," which is described as a noninflammatory lesion. Thoracic aortic aneurysms are a complication of Marfan syndrome and can be inherited in an autosomal dominant manner of familial thoracic aortic aneurysm. METHODS: Full aortic segments were collected from patients undergoing elective repair with Marfan syndrome (n = 5), familial thoracic aortic aneurysm (n = 6), and thoracic aortic aneurysms (n = 9), along with control aortas (n = 5). Immunohistochemistry staining was performed using antibodies directed against markers of lymphocytes and macrophages. Real-time polymerase chain reaction analysis was performed to quantify the expression level of the T-cell receptor beta-chain variable region gene. RESULTS: Immunohistochemistry of thoracic aortic aneurysm aortas demonstrated that the media and adventitia from Marfan syndrome, familial thoracic aortic aneurysm, and sporadic cases had increased numbers of T lymphocytes and macrophages when compared with control aortas. The number of T cells and macrophages in the aortic media of the aneurysm correlated inversely with the patient's age at the time of prophylactic surgical repair of the aorta. T-cell receptor profiling indicated a similar clonal nature of the T cells in the aortic wall in a majority of aneurysms, whether the patient had Marfan syndrome, familial thoracic aortic aneurysm, or sporadic disease. CONCLUSION: These results indicate that the infiltration of inflammatory cells contributes to the pathogenesis of thoracic aortic aneurysms. Superantigen-driven stimulation of T lymphocytes in the aortic tissues of patients with thoracic aortic aneurysms may contribute to the initial immune response.
Subject(s)
Aortic Aneurysm, Abdominal/pathology , Aortic Aneurysm, Thoracic/pathology , Marfan Syndrome/pathology , Receptors, Antigen, T-Cell/immunology , Transforming Growth Factor beta2/immunology , Adult , Aged , Antigens, CD/immunology , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Differentiation, T-Lymphocyte/metabolism , Aortic Aneurysm, Abdominal/immunology , Aortic Aneurysm, Abdominal/surgery , Aortic Aneurysm, Thoracic/immunology , Aortic Aneurysm, Thoracic/surgery , Biomarkers/analysis , Biopsy, Needle , Cardiac Surgical Procedures/methods , Case-Control Studies , Female , Humans , Immunohistochemistry , Male , Marfan Syndrome/genetics , Marfan Syndrome/surgery , Middle Aged , Multivariate Analysis , RNA/analysis , Receptors, Antigen, T-Cell/metabolism , Reference Values , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Statistics, Nonparametric , Tissue Culture Techniques , Transforming Growth Factor beta2/analysis , Vascular Cell Adhesion Molecule-1/immunology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
BACKGROUND: Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK. MK2 is stimulated in a wide range of inflammatory conditions and its catalytic activity is required for cytokine production, cell migration and is a potential drug target for inflammatory diseases. Disruption of MK2 leads to a reduction in TNF-alpha production. MK2-mediated pro-inflammatory cytokine production has been demonstrated in several inflammatory conditions where TNF-alpha plays a role. OBJECTIVE/METHODS: We discuss the development of specific MK2 inhibitors for the treatment of inflammatory diseases. RESULTS/CONCLUSION: Inhibition of the p38 MAPK pathway may have therapeutic uses for inflammatory diseases. However, blocking p38 MAPK activation in vivo is not advisable due to toxicity, significant off-target effects, and lack of oral bioavailability. This concern may be countered by the use of MK2 inhibitors that can dissect the pathways downstream of p38 without affecting additional cellular functions.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Inflammation/drug therapy , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Drug Design , Humans , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
BACKGROUND: The ubiquitin-proteasomal degradation pathway plays a critical role in protein degradation and regulates a wide variety of cellular functions. This highly conserved post-translational modification of proteolytic processes is mainly carried out by substrate-specific E3 ligases. The deregulation of E3 ligases contributes to cancer development and their overexpression is often associated with poor prognosis. OBJECTIVES: We review the current understanding of E3 ligases, their functional role in cancer pathogenesis, current progress and development of certain ubiquitin E3 ligases as targets for therapeutic intervention. METHODS: Preclinical and clinical data for E3 ligase inhibitors available in the public domain are discussed. CONCLUSIONS: With the growing understanding of their role in cancer development and progression, E3 ligases have emerged as potential anticancer targets for therapeutic intervention.
Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Ubiquitin-Protein Ligases/drug effects , Animals , Clinical Trials as Topic , Drug Delivery Systems , Drug Evaluation, Preclinical , Gene Expression Regulation/drug effects , Humans , Neoplasms/enzymology , Prognosis , Protein Processing, Post-Translational/physiology , Ubiquitin-Protein Ligases/metabolismABSTRACT
Human cytomegalovirus (HCMV) infection results in endothelial dysfunction, typically known as dysregulated apoptosis, and aberrant expression and sub-cellular localization of p53, a tumor suppressor that accumulates at the late stage of infection. In this study, we examined three hypotheses that could be responsible for HCMV-induced cytoplasmic p53 accumulation at the later stage of infection: hyperactive nuclear export, cytoplasmic p53 tethering and delayed p53 degradation. Leptomycin B treatment, a nuclear export inhibitor, was unable to reduce cytoplasmic p53, thereby eliminating the hyperactive nuclear export mechanism. The findings that nascent p53 still entered nuclei after the nuclear export inhibition indicated that cytoplasmic tethering may play a minor role. Cytoplasmic p53 was still observed after the translation activities were blocked by cycloheximide. There was more than an eight-fold increase in the cytoplasmic p53 half-life with abnormal p53 ubiquitination. Taken together, these results suggest that delayed degradation could be responsible for the cytoplasmic p53 accumulation. The general slow-down of the proteasomal activity and the dysregulated p53 ubiquitination process at the later stage of infection could contribute to the reduced cytoplasmic p53 degradation and might be relevant to dysregulated endothelial apoptosis. The HCMV-induced changes in p53 dynamics could contribute to endothelial dysfunction.
Subject(s)
Cytomegalovirus Infections/virology , Cytomegalovirus/physiology , Cytoplasm/metabolism , Endothelial Cells/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Apoptosis/physiology , Cell Nucleus/metabolism , Cell Nucleus/virology , Cells, Cultured , Cycloheximide/pharmacology , Cytoplasm/virology , Endothelial Cells/virology , Humans , Karyopherins/biosynthesis , Proto-Oncogene Proteins c-mdm2/drug effects , Proto-Oncogene Proteins c-mdm2/metabolism , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/biosynthesis , Time Factors , Tumor Suppressor Protein p53/drug effects , Ubiquitin/metabolism , Exportin 1 ProteinABSTRACT
ANG II regulates growth factor expression in the kidney. We investigated whether ANG II regulated vascular endothelial growth factor (VEGF) synthesis in proximal tubular epithelial (MCT) cells. ANG II (1 nM) increased VEGF protein expression within 5 min, the effect lasting for 30 min. There was no change in VEGF mRNA levels or mRNA stability, and transcription inhibitors did not affect ANG II-induced VEGF expression. Regulation of VEGF translation was investigated. Polyribosomal analysis revealed selective enrichment of heavy ribosomes (polysomes) with VEGF mRNA transcripts compared with light ribosomes in ANG II-treated cells, although distribution of GAPDH was unaltered. In vitro translation of total RNA from polysomal fractions showed selective increase in VEGF protein synthesis in ANG II-treated cells. Preincubation with LY-294002, a PI 3-kinase inhibitor, or expression of dominant-negative Akt prevented ANG II-stimulated increase in VEGF translation. ANG II increased phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1, critical events that regulate the initiation phase of protein translation. ANG II failed to increase VEGF mRNA translation in cells stably expressing the phosphorylation mutant of 4E-BP1. Our data illustrate that a rapid increase in VEGF protein expression by ANG II is regulated at the initiation phase of translation of VEGF mRNA in renal epithelial cells. Regulation of VEGF translation by ANG II represents a novel pathway of renal response to injury.
Subject(s)
Angiotensin II/pharmacology , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Kidney/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Angiotensin II/antagonists & inhibitors , Animals , Blotting, Northern , Cells, Cultured , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Immunohistochemistry , Kidney/drug effects , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Mice , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Ribosomes/drug effects , Ribosomes/metabolismABSTRACT
The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.
Subject(s)
Endothelial Cells/drug effects , Endothelial Cells/metabolism , Glucose/pharmacology , Heparan Sulfate Proteoglycans/metabolism , Kidney Glomerulus/cytology , Animals , Basement Membrane/drug effects , Basement Membrane/metabolism , Cattle , Cells, Cultured , Diabetic Nephropathies/metabolism , Endothelial Cells/cytology , Gene Expression/drug effects , Heparan Sulfate Proteoglycans/genetics , Sulfur RadioisotopesABSTRACT
In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-beta promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-beta Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-beta Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-beta and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.