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At least 1,117 records · Page 62Linked to original sources
PubMed2006

Thrombin/thrombin receptor (PAR-1)-mediated induction of IL-8 and VEGF expression in prostate cancer cells.

Interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) are two cytokines promoting prostate tumor growth and angiogenesis. The main coagulation protease thrombin may modulate the malignant phenotype of prostate cancer cells via its cellular receptor(s). We aimed to investigate the effects of thrombin on IL-8 and VEGF expression in DU 145 prostate cancer cells. Thrombin induced the expression and secretion of IL-8 and VEGF, with more pronounced effects on IL-8. Target-specific siRNA-induced protease-activated receptor 1 (PAR-1) knockdown completely neutralized thrombin-enhanced cytokine secretion, demonstrating the essential role of PAR-1. Inhibitors of either extracellular signal-regulating kinase (ERK) or phosphatidylinositol 3-kinase (PI3K) partly reversed the thrombin-induced cytokine expression, suggesting that both ERK and PI3K kinase pathways may be involved in IL-8 and VEGF expression. The results suggest that the thrombin/PAR-1 system upregulates cytokines in prostate cancer cells which in turn may contribute to the progression of prostate cancer.

Extracellular Signal-Regulated MAP Kinases↗
PubMed2004

Antimetastatic effect of prodigiosin through inhibition of tumor invasion.

Prodigiosin, a bacterial metabolite, was reported to have immunosuppressive and anticancer activities. In this study, we investigated novel functions of prodigiosin about anti-metastasis and anti-invasion. Prodigiosin dose-dependently inhibited 95-D cells' migration and invasion according to wound healing assay and the Transwell assay. The inhibitive effect could reach about 50% when cells were treated with 5 microM prodigiosin for 12 h. In animal experiment, intraperitoneal administration of 5 mg kg(-1) prodigiosin decreased the number of metastatic nodules by 53% and elevated the survival rate of mice about one-fold comparing with control group. Results of cell aggregation and adhesion assay showed that prodigiosin could promote cell aggregation and simultaneously inhibit cell from adhering to extracellular matrix (ECM). In addition, prodigiosin suppressed RhoA gene expression, hence, decreased protein level of RhoA in 95-D cells, according to RT-PCR assay and Western blot assay. Gel zymogram assay revealed that prodigiosin could suppress the activity of matrix metalloproteinase-2 (MMP-2). These results demonstrate that prodigiosin effectively inhibit tumor metastasis in vitro and in vivo. The action mechanisms of prodigiosin are associated with the promotion of cell aggregation and the inhibition of various steps in cell invasive process, which include the inhibition of cell adhesion and mobility in a RhoA-dependent way and the suppression of MMP-2 ability.

Animals↗
PubMed2006

Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells.

Optimedin, also known as olfactomedin 3, belongs to a family of olfactomedin domain-containing proteins. It is expressed in neural tissues and Pax6 is involved in the regulation of its promoter. To study possible effects of optimedin on the differentiation of neural cells, we produced stably transfected PC12 cell lines expressing optimedin under a tetracycline-inducible promoter. Cells expressing high levels of optimedin showed higher growth rates and stronger adhesion to the collagen extracellular matrix as compared with control PC12 cells. After stimulation with nerve growth factor (NGF), optimedin-expressing cells demonstrated elevated levels of N-cadherin, beta-catenin, alpha-catenin and occludin as compared with stimulated, control PC12 cells. Expression of optimedin induced Ca(2+)-dependent aggregation of NGF-stimulated PC12 cells and this aggregation was blocked by the expression of N-cadherin siRNA. Expression of optimedin also changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. We suggest that expression of optimedin stimulates the formation of adherent and tight junctions on the cell surface and this may play an important role in the differentiation of the brain and retina through the modulation of cytoskeleton organization, cell-cell adhesion and migration.

Animals↗
PubMed1999

Increase in nucleoli after x-radiation of fibroblasts of patients with Gorlin syndrome.

Gorlin syndrome (GS) is an autosomal dominant disorder in which patients are abnormally susceptible to ionizing radiation with radiotherapeutic doses. Radiogenic basal cell carcinomas may develop with a short latent period in patients. The mechanisms underlying the abnormal radiosusceptibility of cells in patients with GS has not been well characterized. In this study we report an increase in the number of nucleoli in fibroblast cells from 3 patients with GS after x-radiation. In GS fibroblasts, the increase in nucleolus number concomitant with the increase of ribonucleoprotein immunoreactive aggregates within the nucleus was observed after x-radiation, whereas significant change was not found in normal fibroblasts derived from healthy donors. This increase disappeared when cells were cultured with the RNA synthesis inhibitor actinomycin D after x-radiation but not when they were cultured with cycloheximide or aphydicolin, which are protein and DNA synthesis inhibitors, respectively. Ultraviolet exposure did not induce remarkable changes in the GS nucleoli. Thus the increase in nucleoli was induced after x-radiation of GS fibroblasts, and this increase seemed to be related to RNA synthesis metabolism.

Basal Cell Nevus Syndrome↗
PubMed2005

Methods for the functional genomic analysis of ubiquitin ligases.

Ubiquitin ligases (E3s) are critical components of the ubiquitin-proteasome system as they are the major determinants of specificity in ubiquitin conjugation. The number of predicted E3s in the mammalian genome is exceeding 400 and is represented by two major subfamilies: HECT domain-containing E3s and RING finger-type E3s. Given the size of this protein family and lack of knowledge on the functions of most of these 400 proteins, their functional annotation should benefit from modern genomic tools. This article presents a methodology consisting of the use of a cDNA expression library to identify suppressors of polyglutamine (polyQ)-mediated protein aggregate formation in cells, as an example of a genomic approach to assign functions to E3s. In this screen, we identified novel RING finger-type E3s exhibiting suppressor activity among >50% of all the potential E3s in the mouse and human genomes. This method could be adapted easily to identify E3s that function in other processes and signaling pathways.

Algorithms↗
PubMed1995

Expression of N-cadherin and alpha-catenin in astrocytomas and glioblastomas.

We examined levels of mRNA and protein for N-cadherin, the predominant cadherin in neural tissues, and mRNA levels for the cadherin-associated protein, alpha-catenin, in a series of gliomas and in glioblastoma cell lines. mRNA levels for N-cadherin and alpha-catenin were significantly higher in glioblastomas than in low-grade astrocytomas or normal brain, while the levels of intact N-cadherin protein were similar in glioblastomas, low-grade astrocytomas and brain. In addition, there was no consistent relationship between invasiveness and expression of N-cadherin and alpha-catenin in highly invasive vs minimally invasive tumours within the same histopathological grade. To assess further the relationship between cadherin expression and neural tumour invasion, we measured N-cadherin expression, calcium-dependent cell adhesion and motility of several glioblastoma cell lines. While all N-cadherin-expressing lines were adhesive, no correlation was seen between the level of N-cadherin expression and cell motility. Together, these findings imply that, in contrast to the role played by E-cadherin in carcinomas, N-cadherin does not restrict the invasion of glioblastomas.

Astrocytoma↗
PubMed2001

Synaptic regulation of protein synthesis and the fragile X protein.

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use "synaptoneurosomes," a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

Animals↗
PubMed2002

The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase.

Rice (Oryza sativa) accumulates prolamins and glutelins as storage proteins. The latter storage protein is synthesized on the endoplasmic reticulum (ER) as a 57-kD proglutelin precursor, which is then processed into acidic and basic subunits in the protein storage vacuole. Three esp2 mutants, CM1787, EM44, and EM747, contain larger amounts of the 57-kD polypeptide and corresponding lower levels of acidic and basic glutelin subunits than normal. Electron microscopic observation revealed that esp2 contained normal-appearing glutelin-containing protein bodies (PB-II), but lacked the normal prolamin-containing PB (PB-I). Instead, numerous small ER-derived PBs of uniform size (0.5 microm in diameter) and low electron density were readily observed. Immunoblot analysis of purified subcellular fractions and immunocytochemistry at the electron microscopy level showed that these new PBs contained the 57-kD proglutelin precursor and prolamin polypeptides. The 57-kD proglutelin was extracted with 1% (v/v) lactic acid solution only after removal of cysteine-rich prolamin polypeptides, suggesting that these proteins form glutelin-prolamin aggregates via interchain disulfide bonds within the ER lumen. The endosperm of esp2 mutants contains the lumenal chaperones, binding protein and calnexin, but lacks protein disulfide isomerase (PDI) at the protein and RNA levels. The transcript of PDI was expressed in the seed only during the early stage of seed development in the wild type. These results suggest that PDI plays an essential role in the segregation of proglutelin and prolamin polypeptides within the ER lumen.

Blotting, Northern↗
PubMed1995

A chromomeric model for nuclear and chromosome structure.

The basic structural elements of chromatin and chromosomes are reviewed. Then a model involving only three architectural motifs, nucleosomes, chromatin loops and transcription factories/chromomeres, is presented. Loops are tied through transcription factors and RNA polymerases to factories during interphase and to the remnants of those factories, chromomeres, during mitosis. On entry into mitosis, increased adhesiveness between nucleosomes and between factories drives a 'sticky-end' aggregation to the most compact and stable structure, a cylinder of nucleosomes around an axial chromomeric core.

Cell Nucleolus↗
PubMed2000

Combined defect in membrane expression and activation of platelet GPIIb--IIIa complex without primary sequence abnormalities in myeloproliferative disease.

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

Increased endothelin-1 content in the platelets of hemodialysed patients.

Patients undergoing hemodialysis, present platelets (PLTs) with physiological dysfunction. Aggregated PLTs stimulate endothelin-1 (ET-1) secretion from endothelial cells. In turn, ET-1 abolishes PLT aggregation. The aim of this study was to determine any presence of ET-1 in the PLTs of hemodialysed patients and to compare its levels with normal subjects. Platelets, isolated from hemodialysed patients, revealed lower aggregation (76 +/- 13%) compared with healthy subjects (96 +/- 2%). Plasma ET-1 was increased in hemodialysed patients 23.5 +/- 3.2 fmol/ml, vs. 10.9 +/- 1.6 fmol/ml in normal subjects. Immunoreactive endothelin-1 was detected in the platelets of both groups. The intraplatelet ET-1 of hemodialysed patients was 13.8 +/- 3.1 fmol/mg protein, vs. 7.9 +/- 1.3 fmol/mg protein in normal individuals. Total RNA was isolated from platelets and reverse transcriptase-mediated polymerase chain reaction (RT-PCR) revealed no presence of the preproET-1 mRNA in either normal or hemodialysed platelets. This suggests that ET-1 is internalized from the plasma. In summary, platelets contain but do not express ET-1. The increased levels of ET-1 in the platelets of the hemodialysed patients may be partly responsible for their lower aggregation.

Adult↗
PubMed2001

Role of N-cadherin and protein kinase C in osteoblast gene activation induced by the S252W fibroblast growth factor receptor 2 mutation in Apert craniosynostosis.

Apert (Ap) syndrome is characterized by premature cranial suture ossification caused by fibroblast growth factor receptor 2 (FGFR-2) mutations. We studied the role of cadherins and signaling events in the phenotypic alterations induced by the Ap FGFR-2 S252W mutation in mutant immortalized fetal human calvaria osteoblasts. The FGFR-2 mutation caused increased expression of the osteoblast markers alkaline phosphatase (ALP), type 1 collagen (COLIA1), and osteocalcin (OC) in long-term culture. The mutation also increased cell-cell aggregation, which was suppressed by specific neutralizing anti-N- and anti-E-cadherin antibodies. Mutant osteoblasts showed increased N- and E-cadherin, but not N-cell adhesion molecule (N-CAM) messenger RNA (mRNA) and protein levels. This was confirmed in vivo by the abundant immunoreactive N- and E-cadherins in preosteoblasts in the Ap suture whereas N-CAM and alpha- and beta-catenins were unaffected. Neutralizing anti-N-cadherin antibody or N-cadherin antisense (AS) oligonucleotides but not anti-E-cadherin antibody or AS reduced ALP activity as well as ALP, COLIA1, and OC mRNA overexpression in mutant osteoblasts. Analysis of signal transduction revealed increased phospholipase Cgamma (PLCgamma) and protein kinase Calpha (PKCalpha) phosphorylation and increased PKC activity in mutant cells in basal conditions. Inhibition of PKC by calphostin C or the PKCalpha-specific inhibitor Gö6976 suppressed the increased N-cadherin mRNA and protein levels as well as the overexpression of ALP, COLIA1, and OC mRNA in mutant cells. Thus, N-cadherin plays a role in the activation of osteoblast differentiation marker genes in mutant osteoblasts and PKCalpha signaling appears to be involved in the increased N-cadherin and osteoblast gene expression induced by the S252W FGFR-2 mutation in human osteoblasts.

Acrocephalosyndactylia↗
PubMed1987

Multiple mutations involved in the phenotype of a temperature-sensitive small-plaque mutant of poliovirus.

A temperature-sensitive small-plaque mutant of poliovirus type 1, ts247, has been analyzed previously. Several mutations were detected in the P3 region of the genome by analysis of proteins and by T1 oligonucleotide mapping of viral RNA. We have now studied spontaneous reversion of ts247 to the wild-type phenotype. This was found to be a two-step event, reversion to a ts+ phenotype (revertant R247-51) being distinct from acquisition of normal plaque size (revertant R247-12). The mutation responsible for the ts phenotype of ts247, implicated also in virus aggregation and heat lability, could not be detected by biochemical studies. Analysis of homotypic recombinants obtained by crossing ts247 with a guanidine-resistant derivative of a temperature-sensitive replicase mutant mapped this mutation to the P1 region or to the 5' end of the P2 region of the genome. The small-plaque phenotype of ts247 and R247-51 was correlated with an abnormality in polypeptide 3C (protease); direct sequencing of viral RNA revealed a U to C change at nucleotide 5658, which altered an isoleucine to threonine in the protease of ts247 and R247-51 but not of R247-12. Two other mutations were present in the region of the genome coding for polypeptide 3D of ts247 and of both classes of revertants. They thus seemed to play no role in the phenotype of ts247. One mutation, an A to G change at nucleotide 7135, was silent at the protein level, whereas the other, an A to G change at nucleotide 6264, determined a major amino acid change from glutamate to glycine in the viral replicase.

Genes↗
PubMed2002

[The sentinel lymph node in melanoma: utilization of molecular biology (RT-PCR) to detect occult metastases].

Sentinel lymph node (SLN) analysis allows the detection of occult metastases in patients with melanoma. The use of serial sections and immunohistochemical investigations (ICH) increases the chance of identifying metastases. Nevertheless, detection of mRNA of the tyrosinase gene through reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive tool for detection of occult melanoma cells in SLN. From September 1999 to August 2001, in the Anatomic Pathology Unit of M. Bufalini Hospital of Cesena, 489 SLNs from 332 patients with primary melanoma in clinical stages I and II (according to AJCC) were examined. There were 66 (13.5%) SLNs and 58 (17.4%) cases with metastasis revealed by histology and ICH. A single case with metastatic SLN was found in patients with melanomas < or = 1 mm in thickness. The percentage of cases with metastases in SLN correlated with thickness of the primary melanoma (p < 0.0001). RT-PCR for tyrosinase was carried out in 448 SLNs from of 308 cases. Overall, the RT-PCR results were positive in 149 (48.4%) patients and in 169 SLNs (37.9%). RT-PCR results showed a strong positive correlation with tumor thickness of primary melanoma (p < 0.0001) and with the clinical stage (p < 0.0001). Of the RT-PCR-positive cases, 18 showed intracapsular aggregates of nevus cells. Besides the percentage of positive cases, once those with nevus aggregates were excluded, overall, the RT-PCR revealed the presence of tyrosinase mRNA in 34.5% of patients with negative histology and ICH. Ongoing monitoring is necessary to define the real prognostic implication of the presumed presence of occult melanoma deposits disclosed by RT-PCR in SLNs.

Adult↗
PubMed1980

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.

Base Sequence↗