ABSTRACT
High-yield dairy cows need high energy feed during periods of increased milk production. The transitional feeding to high energy feed increases the risk of developing a variety of metabolic disorders. Here, five Holstein cows were fed a four-stage feeding protocol (3 weeks for each stage) ranging from 54.9 to 73.7% total digestive nutrients (TDN). The purpose of the study was to investigate the effect of lactic acid bacteria on high-energy-fed cows associated with transitional feeding, and to evaluate the effects of probiotics on intestinal bacterial changes and inflammatory responses. Three feed transition periods were established for five cows, and Lactobacillus plantarum RGU-LP1 (LP1) was fed as a probiotic during the high-energy feeding period. The number of lymphocyte subsets such as CD3-, CD4-, and CD8 positive cells decreased in response to the high energy feed. Lipopolysaccharide (LPS)-induced cytokine (IL-1ß and IL-2) gene expression in peripheral blood mononuclear cells (PBMCs) was shown to increase in those animals receiving the high energy feed. However, supplementation with LP1 resulted in an increase in the number of lymphocyte subsets and the expression of IL-1ß and IL-2 were returned to the level at low energy diet. These results suggest that high energy diets induce inflammatory cytokine responses following LPS stimulation, and that the addition of LP1 mitigates these results by regulating the LPS-induced inflammatory reaction. Therefore, the functional lactic acid bacteria LP1 is expected to regulate inflammation resulting from high energy feeding, and this probiotic could be applied to support inflammatory regulation in high-yield dairy cows.
ABSTRACT
BACKGROUND: Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs. METHODS: Neurospheres and CD133⺠cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines. RESULTS: Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⺠cells compared with CD133â» cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. CONCLUSION: Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.
Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Integrin alpha3/genetics , Integrin alpha3/physiology , Neoplastic Stem Cells/metabolism , Brain Neoplasms/genetics , Cell Adhesion/genetics , Cell Movement/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibronectins/metabolism , Gene Expression Regulation, Neoplastic , Glioma/genetics , Humans , Integrin alpha3/metabolism , MAP Kinase Signaling System/physiology , Neoplasm Invasiveness , Neoplastic Stem Cells/pathology , Phosphorylation , Tumor Cells, Cultured , Up-Regulation/geneticsABSTRACT
Using expression cloning to screen a human fetal kidney cDNA library for regulator(s) of pro-matrix metalloproteinase (MMP)-2 processing mediated by membrane-type (MT) 1 MMP, we isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes the NH(2)-terminal 313-amino acid region of a calcium-binding proteoglycan, testican 3, with a 3-amino acid substitution at the COOH terminus and thus was named N-Tes. N-Tes comprises a signal peptide, a unique domain, a follistatin-like domain, and a Ca(2+)-binding domain but lacks a COOH-terminal thyroglobulin domain and two putative glycosaminoglycan attachment sites of testican 3. Pro-MMP-2 activation by MT3-MMP was also inhibited by the coexpression of N-Tes. Immunoprecipitation analysis demonstrated direct interaction of N-Tes with either MT1-MMP or MT3-MMP. Expression of testican 1 or testican 3 but not testican 2 also inhibited pro-MMP-2 activation by either MT1-MMP or MT3-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that the unique NH(2)-terminal domain of N-Tes is responsible for the inhibition of pro-MMP-2 activation by MT-MMPs. Expression of N-Tes and testican 3 was detected in normal brain but down-regulated in glioma tissues. Transfection of either the N-Tes or testican 3 gene into U251 glioma cells or Madin-Darby canine kidney cells transformed by erbB2 suppressed their invasive growth in collagen gel. These results suggest that both N-Tes and testican 3 would interfere with tumor invasion by inhibiting MT-MMPs.
Subject(s)
Enzyme Precursors/antagonists & inhibitors , Gelatinases/antagonists & inhibitors , Metalloendopeptidases/antagonists & inhibitors , Proteoglycans/physiology , Alternative Splicing , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/genetics , Dogs , Down-Regulation , Enzyme Activation , Enzyme Precursors/metabolism , Gelatinases/metabolism , Gene Library , Glioma/enzymology , Glioma/genetics , Glioma/pathology , Humans , Kidney/cytology , Kidney/physiology , Matrix Metalloproteinase 16 , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/metabolism , Molecular Sequence Data , Peptide Mapping , Protein Isoforms , Protein Sorting Signals/genetics , Protein Structure, Tertiary , Proteoglycans/biosynthesis , Proteoglycans/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transfection , Tumor Cells, CulturedABSTRACT
Ets transcription factors are associated with tumor malignancy. We reported previously that the stable transfection of the dominant-negative form of Ets-1 (Ets-DN) in the glioma cell line U251 induced down-regulation of urokinase-type plasminogen activator mRNA expression and invasiveness (M. Nakada et al., J. Neuropathol. Exp. Neurol., 58: 329-334, 1999). Here we analyzed effects of Ets-DN expression on cell adhesion, migration, and phosphorylation of focal adhesion kinase. U251 cells expressing Ets-DN (U251-DN) showed reduced cell adhesion, spreading, and extension of actin stress fibers on dishes coated with fibronectin but not on dishes coated with collagen. Migration of U251-DN cells was found to be significantly inhibited compared with that of parental cells when examined by wound-induced migration assay on fibronectin-coated dishes. Phosphorylation levels of focal adhesion kinase in U251-DN cells were also attenuated on dishes coated with fibronectin. Reduced expression level of integrin alpha5 subunit in U251-DN cells was demonstrated by semiquantitative reverse transcription-PCR analysis. Semiquantitative reverse transcription-PCR of surgical samples of brain tumors revealed that the expression level of Ets-1 mRNA correlated with that of integrin alpha5 mRNA in glioma. The experimental metastatic ability of U251-DN cells examined in chick embryo was considerably lower than that of parental cells. These results suggest that Ets-1 contributes to glioma malignancy by up- regulating expression of the integrin alpha5 subunit, which composes integrin alpha5beta1 and mediates intracellular signaling and the subsequent acceleration of the invasive process, including cell adhesion and migration.
Subject(s)
Antigens, CD/biosynthesis , Fibronectins/pharmacology , Glioma/pathology , Proto-Oncogene Proteins/biosynthesis , Transcription Factors/biosynthesis , Animals , Antigens, CD/genetics , Cell Adhesion , Cell Movement , Chick Embryo , Collagen Type I/metabolism , Collagen Type I/pharmacology , Down-Regulation , Fibronectins/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/metabolism , Globins/genetics , Humans , Integrin alpha5 , Integrin beta3 , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Phosphorylation , Platelet Membrane Glycoproteins/biosynthesis , Platelet Membrane Glycoproteins/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-ets , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transcription Factors/genetics , Transcription Factors/physiology , Transfection , Tumor Cells, CulturedABSTRACT
Carperitide (synthetic atrial natriuretic peptide) is a newly developed drug for the treatment of heart failure. Although this drug has been used for various types of heart failure, it remains unknown whether it has additive effects on hemodynamic parameters or renal excretory function during intensive treatment for acute refractory heart failure. We have examined the cardiorenal and hormonal effects of carperitide (0.05-0.10 microg / min / kg) in 9 patients (mean age: 67+/-8 years) with severe heart failure complicated with acute myocardial infarction, in which a range of intensive treatments have already been started. Hemodynamic parameters were determined before and 4, 24 and 48 hours after initiation of carperitide. Pulmonary capillary wedge pressure (mean+/-SD) had decreased dramatically from 21+/-6 to 11+/-5 mmHg (p<0.01) 4 hours after the treatment without significant renal effects. Heart rate and systemic blood pressure were not significantly changed. These beneficial effects were maintained for at least 24 hours. Plasma aldosterone levels fell significantly in response to the drug (from 148+/-68 to 56+/-29 pg / ml; p<0.05). However, mean hourly urine output remained unchanged after carperitide. In conclusion, intravenous infusion of carperitide promptly and persistently reduces left ventricular filling pressure without diuresis, hypotension, reflex tachycardia, or neurohormonal activation in patients with refractory heart failure due to severe acute myocardial infarction.
Subject(s)
Atrial Natriuretic Factor/therapeutic use , Bronchodilator Agents/therapeutic use , Heart Failure/drug therapy , Myocardial Infarction/drug therapy , Peptide Fragments/therapeutic use , Aged , Aldosterone/blood , Blood Pressure/drug effects , Female , Heart Failure/etiology , Heart Failure/physiopathology , Heart Rate/drug effects , Humans , Infusions, Intravenous , Male , Middle Aged , Myocardial Infarction/complications , Myocardial Infarction/physiopathology , Renin/blood , Vascular Resistance/drug effectsABSTRACT
Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.
Subject(s)
Cell Membrane/enzymology , Matrix Metalloproteinase 2/metabolism , Membrane Proteins/chemistry , Membrane Proteins/physiology , Metalloendopeptidases/metabolism , Animals , COS Cells , Cell Line , Claudin-1 , Claudin-3 , Claudin-5 , Claudins , Cloning, Molecular , DNA, Complementary/metabolism , Enzyme Activation , Enzyme-Linked Immunosorbent Assay , Gene Deletion , Gene Library , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Matrix Metalloproteinase 14 , Matrix Metalloproteinases, Membrane-Associated , Membrane Proteins/metabolism , Microscopy, Fluorescence , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Tight Junctions , Tissue Inhibitor of Metalloproteinase-2/metabolism , TransfectionABSTRACT
A new vanadyl complex, bis(5-iodopicolinato)oxovanadium(IV), VO(IPA)2, with a VO(N2O2) coordination mode, was prepared by mixing 5-iodopicolinic acid and VOSO4 at pH 5, with the structure characterized by electronic absorption, IR, and EPR spectra. Introduction of the halogen atom on to the ligand enhanced the in vitro insulinomimetic activity (IC50 = 0.45 mM) compared with that of bis(picolinato)oxovanadium(IV) (IC50 = 0.59 mM). The hyperglycemia of streptozotocin-induced insulin-dependent diabetic rats was normalized when VO(IPA)2 was given by daily intraperitoneal injection. The normoglycemic effect continued for more than 14 days after the end of treatment. To understand the insulinomimetic action of VO(IPA)2, the organ distribution of vanadium and the blood disposition of vanadyl species were investigated. In diabetic rats treated with VO(IPA)2, vanadium was distributed in almost all tissues examined, especially in bone, indicating that the action of vanadium is not peripheral. Vanadyl concentrations in the blood of normal rats given VO(IPA)2 remain significantly higher and longer than those given other complexes because of its slower clearance rate. VO(IPA)2 binds with the membrane of erythrocytes, probably owing to its high hydrophobicity in addition to its binding with serum albumin. The longer residence of vanadyl species shows the higher normoglyceric effects of VO(IPA)2 among three complexes with the VO(N2O2) coordination mode. On the basis of these results, VO(IPA)2 is indicated to be a preferred agent to treat insulin-dependent diabetes mellitus in experimental animals.
Subject(s)
Hypoglycemic Agents/chemical synthesis , Organometallic Compounds/pharmacology , Vanadates/pharmacokinetics , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Animals , Blood Circulation , Blood Glucose/drug effects , Diabetes Mellitus, Experimental/drug therapy , Drug Evaluation, Preclinical , Erythrocytes/metabolism , Fatty Acids, Nonesterified/antagonists & inhibitors , Fatty Acids, Nonesterified/metabolism , Hypoglycemic Agents/pharmacokinetics , Hypoglycemic Agents/pharmacology , Inhibitory Concentration 50 , Iodine/chemistry , Ligands , Metabolic Clearance Rate , Organometallic Compounds/chemical synthesis , Picolinic Acids/chemical synthesis , Picolinic Acids/pharmacokinetics , Picolinic Acids/pharmacology , Rats , Rats, Wistar , Spectrum Analysis , Streptozocin , Tissue Distribution , Vanadates/chemical synthesis , Vanadates/chemistry , Vanadates/pharmacologyABSTRACT
According to Pearson's HSAB (hard and soft acids and bases) rule, the vanadyl ion is classified as a hard acid. However, vanadyl-cysteine methyl ester and dithiocarbamate complexes with VO(S2N2) and VO(S4) coordination modes, respectively, that contain bonds with a combination of hard acid (VO2+) and soft base (sulfur) have been found to form stable complexes and exhibit insulin-mimetic activities in in vitro and in vivo evaluations. Based on such observations, a purple bis(1-oxy-2-pyridinethiolato)oxovanadium(IV) (VO(OPT)) complex with VO(S2O2) coordination mode was prepared and found to have a strong insulin-mimetic activity in in vitro evaluation, which followed in vivo effectiveness on intraperitoneal injection and oral administration. Then, we examined the real-time ESR analysis of vanadyl species in the blood of live rats given VO(OPT) by use of the blood circulation monitoring-ESR method. The clearance of vanadyl species from the blood in terms of half-life (t(1/2)) was determined as 15 min in VO(OPT)-treated rats, while t(1/2) of VOSO4-treated rats was 5 min, indicating the long-term acting character of VO(OPT). On the basis of the results, VO(OPT) with VO(S2O2) coordination mode is proposed to be a potent orally active insulin-mimetic complex in treating insulin-dependent diabetes mellitus in experimental animals.
Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus, Experimental/drug therapy , Hypoglycemic Agents/pharmacology , Vanadates/pharmacology , Adipocytes/drug effects , Adipocytes/metabolism , Administration, Oral , Animals , Body Weight/drug effects , Diabetes Mellitus, Experimental/blood , Dose-Response Relationship, Drug , Drug Evaluation , Electron Spin Resonance Spectroscopy , Fatty Acids, Nonesterified/metabolism , Half-Life , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacokinetics , Injections, Intravenous , Insulin/pharmacology , Male , Molecular Structure , Rats , Rats, Wistar , Vanadates/chemistry , Vanadates/pharmacokineticsABSTRACT
Transfection of the mouse membrane type-2 matrix metalloproteinase (MT2-MMP) gene into COS-1 cells resulted in activation of progelatinase A; however, that of the human gene had no effect. Expression of human and mouse MT2-MMP chimeric proteins revealed the defect of human MT2-MMP which resides in the region between amino acid (aa) residues 155 and 271. Seven aa residues in this region were not conserved between human and mouse MT2-MMP. Substitution with the corresponding mouse residue, proline-183 to serine and glutamine-185 to aspartic acid, recovered cell-associated progelatinase A activation function. These residues are located in the insertion sequence-2 (IS-2), which was conserved in six clones of the human MT2-MMP gene from different sources, except that of proline-183 which was substituted with serine from HT1080 cells. These results indicate that human MT2-MMP is defective in cell-associated activation of progelatinase A, and this is attributed to IS-2. These findings emphasize the importance of IS-2 in MT2-MMP functionality.
Subject(s)
Enzyme Precursors/metabolism , Gelatinases/metabolism , Metalloendopeptidases/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Blotting, Western , COS Cells , Cell Membrane/enzymology , DNA Transposable Elements , Enzyme Activation , Enzyme Precursors/genetics , Gelatinases/genetics , Humans , Matrix Metalloproteinase 15 , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Mice , Molecular Sequence Data , Mutagenesis, Insertional , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein Processing, Post-Translational , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , TransfectionABSTRACT
The mechanisms of the lymph node metastasis remain unclear. We demonstrate the role of MT1-MMP on the lymph node metastasis using in vivo experimental model of lymph node metastasis by orthotopic implantation of MT1-MMP transfected gastric cancer cell line in the stomach of nude rats. TMK-1 cell line without expression of MT1-MMP was transfected with the pcDNA3 plasmid containing a 3.4-kb MT1-MMP cDNA fragment by calcium phosphate method, and the transfected cell line was designated as TMK-MT. Western blot and RT-PCR analyses showed the specific bands corresponding to MT1-MMP in the TMK-MT cells. By gelatin zymography, the activated form (62-kDa) of MMP-2 was identified in the medium of TMK-MT cell line, but was not detected in TMK-1 cells. Six weeks after orthotopic implantation of TMK-1 and TMK-MT xenografts of nude mouse-subcutaneus tumor into the stomach of nude rats, gastric tumors were found in all the animals. Histologically, the lymphatic invasion was found in the submucosa of the TMK-MT gastric tumors. Lymph node metastasis was not detected in nude rats bearing TMK-1 gastric tumor (0/8). In contrast, lymph node metastasis was detected in five out of 8 rats, bearing TMK-MT gastric tumor. MT1-MMP immunoreactivity was found on the cell membrane and cytoplasm of TMK-MT cells not only in the lymph node metastasis but also in the stomach tumor. These results suggest that MT1-MMP overexpression induced by transfection of its gene may promote lymph node metastasis of transformed cells.
Subject(s)
Metalloendopeptidases/physiology , Neoplasm Proteins/physiology , Stomach Neoplasms/pathology , Animals , Female , Lymphatic Metastasis , Matrix Metalloproteinase 14 , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinases, Membrane-Associated , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Rats , Rats, Inbred F344 , Rats, Nude , Specific Pathogen-Free Organisms , TransfectionABSTRACT
Alteration in myocardial collagen metabolism is an important factor in the progression of ventricular remodeling after myocardial infarction (MI). This study examined sequential changes in circulating levels of carboxyterminal propeptide of type I procollagen (PICP) as a collagen synthesis marker in order to assess the value of PICP for predicting the progression of left ventricular remodeling after MI. The study group comprised 20 patients with first MI undergoing reperfusion therapy. Peripheral blood samples were obtained on admission and serially up to 4 weeks after admission. Circulating levels of PICP and B-type natriuretic peptide (BNP), a tentative biochemical marker for the severity of MI, were measured by direct radioimmunoassay. Left ventricular end-diastolic volume index (EDVI) in acute and chronic phases were determined by left ventriculography, and changes (Delta) in EDVI were used as an index of left ventricular remodeling. Plasma PICP levels in the non-dilation group (< median DeltaEDVI) showed no significant change. However, in the dilation group (> median DeltaEDVI) PICP started to increase significantly 3 days after admission, peaking on day 14 (from 74 +/- 6 to 104 +/- 19 ng/ml, p < 0.05). DeltaEDVI was significantly correlated with plasma PICP at 2 and 3 weeks, and with plasma BNP at 1, 2 and 3 weeks. Plasma PICP 2 weeks after MI was the only independent predictor of DeltaEDVI (p < 0.001). These results suggest that an increase in plasma PICP levels 2 weeks after admission is a useful biochemical predictor of the progression of ventricular remodeling after MI.
Subject(s)
Myocardial Infarction/blood , Peptide Fragments/blood , Procollagen/blood , Ventricular Dysfunction, Left/physiopathology , Ventricular Remodeling , Aged , Biomarkers/blood , Female , Humans , Male , Middle Aged , Peptide Fragments/biosynthesis , Predictive Value of Tests , Procollagen/biosynthesis , Prognosis , Regression Analysis , Sensitivity and Specificity , Ventricular Remodeling/physiologyABSTRACT
Cell migration is modulated by regulatory molecules such as growth factors, oncogenes, and the tumor suppressor PTEN. We previously described inhibition of cell migration by PTEN and restoration of motility by focal adhesion kinase (FAK) and p130 Crk-associated substrate (p130(Cas)). We now report a novel pathway regulating random cell motility involving Shc and mitogen-activated protein (MAP) kinase, which is downmodulated by PTEN and additive to a FAK pathway regulating directional migration. Overexpression of Shc or constitutively activated MEK1 in PTEN- reconstituted U87-MG cells stimulated integrin- mediated MAP kinase activation and cell migration. Conversely, overexpression of dominant negative Shc inhibited cell migration; Akt appeared uninvolved. PTEN directly dephosphorylated Shc. The migration induced by FAK or p130(Cas) was directionally persistent and involved extensive organization of actin microfilaments and focal adhesions. In contrast, Shc or MEK1 induced a random type of motility associated with less actin cytoskeletal and focal adhesion organization. These results identify two distinct, additive pathways regulating cell migration that are downregulated by tumor suppressor PTEN: one involves Shc, a MAP kinase pathway, and random migration, whereas the other involves FAK, p130(Cas), more extensive actin cytoskeletal organization, focal contacts, and directionally persistent cell motility. Integration of these pathways provides an intracellular mechanism for regulating the speed and the directionality of cell migration.
Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Cell Adhesion Molecules/metabolism , Cell Movement , Mitogen-Activated Protein Kinase Kinases , Phosphoric Monoester Hydrolases/metabolism , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Tumor Suppressor Proteins , Actins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Adhesion , Cell Adhesion Molecules/genetics , Crk-Associated Substrate Protein , Culture Media, Serum-Free , Cytoskeleton/metabolism , Enzyme Activation , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Genes, Dominant/genetics , Genes, Dominant/physiology , Humans , Integrins/antagonists & inhibitors , Integrins/metabolism , MAP Kinase Kinase 1 , PTEN Phosphohydrolase , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Proteins/antagonists & inhibitors , Proteins/genetics , Retinoblastoma-Like Protein p130 , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , Transfection , Tumor Cells, CulturedABSTRACT
The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.
Subject(s)
Cell Adhesion Molecules/metabolism , Extracellular Matrix/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphoric Monoester Hydrolases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Proteins , Cell Adhesion Molecules/chemistry , Cell Survival , Enzyme Inhibitors/pharmacology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , PTEN Phosphohydrolase , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins c-akt , Tumor Cells, Cultured , Tyrosine/metabolismABSTRACT
PTEN/MMAC1 is a major new tumor suppressor gene that encodes a dual-specificity phosphatase with sequence similarity to the cytoskeletal protein tensin. Recently, we reported that PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits cell migration, spreading, and focal adhesion formation. Here, the effects of PTEN on cell invasion, migration, and growth as well as the involvement of FAK and p130 Crk-associated substrate (p130Cas) were investigated in U87MG glioblastoma cells missing PTEN. Cell invasion, migration, and growth were down-regulated by expression of phosphatase-active forms of PTEN but not by PTEN with an inactive phosphatase domain; these effects were correlated with decreased tyrosine phosphorylation levels of FAK and p130Cas. Overexpression of FAK concomitant with PTEN resulted in increased total tyrosine phosphorylation levels of FAK and p130Cas and effectively antagonized the effects of PTEN on cell invasion and migration and partially on cell growth. Overexpression of p130Cas increased total tyrosine phosphorylation levels of p130Cas without affecting those of FAK; however, although p130Cas could reverse PTEN inhibition of cell invasion and migration, it did not rescue cell growth in U87MG cells. In contrast to FAK, p130Cas could not be shown to interact with PTEN in cells, and it was not dephosphorylated directly by PTEN in vitro. These results suggest important roles of PTEN in the phenotype of tumor progression, and that the effects of PTEN on cell invasion, migration, and growth are mediated by distinct downstream pathways that diverge at the level of FAK.
Subject(s)
Cell Adhesion Molecules/physiology , Genes, Tumor Suppressor , Phosphoproteins/physiology , Phosphoric Monoester Hydrolases/physiology , Protein-Tyrosine Kinases/physiology , Proteins , Tumor Suppressor Proteins , Cell Division , Cell Movement , Crk-Associated Substrate Protein , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Neoplasm Invasiveness , PTEN Phosphohydrolase , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Retinoblastoma-Like Protein p130 , Tumor Cells, CulturedABSTRACT
The purpose of this study was to determine the effectiveness and the limitation of the MR cholangiopancreatography (MRCP) in the initial diagnosis for the biliary and pancreatic diseases compared with the image of Endoscopic retrograde cholangiopancreatography (ERCP). MRCP showed higher performance toward the discovery of the choledocholithiasis of a diameter 4 mm and more. Furthermore, MRCP reflected a malignant tumor at the constrictive part and the dilated proximal part of the biliary and pancreatic duct in 23 example all. An advantage of ERCP was the ability to carry out biopsies and therapeutic procedures, such as biliary drainage and sphincterotomy, while MRCP was an important diagnostic modality in the work-up of the biliary and pancreatic diseases and could help in planning treatment. MRCP would become the examination method first used, and it would be popularized all the more from now on.
Subject(s)
Biliary Tract Diseases/diagnosis , Cholangiopancreatography, Endoscopic Retrograde/methods , Magnetic Resonance Imaging/methods , Pancreatic Diseases/diagnosis , Bile Duct Neoplasms/diagnosis , Gallstones/diagnosis , Gallstones/pathology , Humans , Image Enhancement/methods , Pancreatic Neoplasms/diagnosisABSTRACT
In order to explore the relationship between the pharmacokinetic properties and pharmacological actions of lipophilic drugs injected with lipid carrier systems, probucol was selected as a model drug with high lipophilicity, and the effect of disposition control on cholesterol-lowering activities was evaluated. Both large emulsion, with mean diameter of 280 nm, and long-circulating type small emulsion containing egg sphingomyelin with mean diameter of 100 nm, showed stable incorporation of probucol. The former produced rapid accumulation of probucol in the liver, while the latter demonstrated prolonged systemic circulation and gradual hepatic uptake. On the other hand, injection of a micellar solution with HCO-60 (polyoxyethylene hydrogenated castor oil) showed a rapid decrease in plasma concentration and a high hepatic uptake of probucol, similar to injections with serum, suggesting the rapid release of the drug from the micelles. However, probucol in a micellar solution showed higher cholesterol-lowering action than that in emulsion formulations. These results suggested that the pharmacological action of probucol in the liver might be affected by the uptake mode and sequential disposition in the organ, depending on the drug retention properties of the lipid carrier particles.
Subject(s)
Anticholesteremic Agents/administration & dosage , Anticholesteremic Agents/pharmacology , Probucol/administration & dosage , Probucol/pharmacology , Animals , Anticholesteremic Agents/pharmacokinetics , Chemical Phenomena , Chemistry, Physical , Cholesterol/blood , Drug Carriers , Emulsions , Lipids , Liver/metabolism , Male , Mice , Micelles , Probucol/pharmacokinetics , Tissue DistributionABSTRACT
Thirty-two patients with chronic active hepatitis C were initially treated with 9 million units of interferon (IFN)-alpha 2a daily for 4 weeks and then thrice weekly for 20 weeks. The incidence of development and influence on clinical effectiveness of anti-IFN alpha 2a neutralizing antibodies measured by bioassay were investigated. Thirteen (41%) of the 32 patients developed antibodies, 4 (12.5%) of whom exhibited a high titer of 128 NU or greater. There was a significant difference (p < 0.05) between responders and non-responders in the low viral load group (less than 10(5) copies/50 microliters) in the incidence of antibody development, with antibodies present in 2 of the 9 responders (22%) and 5 of the 7 non-responders (71%). There was also a significant difference between the antibody-negative and antibody-positive patients in circulating HCV-RNA, 2-5AS activity and average ALT levels at the latter half of treatment. In addition, 2-5AS activity was significantly lower (p < 0.05) at completion of treatment in the high antibody titer group (128 NU or greater) than in the low antibody titer group. These results indicate that the development of anti-IFN neutralizing antibodies had a definite influence on the clinical course of IFN treatment for chronic hepatitis C.
Subject(s)
Antibodies/analysis , Hepatitis C/immunology , Hepatitis C/therapy , Hepatitis, Chronic/immunology , Hepatitis, Chronic/therapy , Interferon-alpha/administration & dosage , Interferon-alpha/immunology , Adult , Aged , Drug Administration Schedule , Female , Hepacivirus/genetics , Humans , Interferon alpha-2 , Male , Middle Aged , RNA, Viral/blood , Recombinant ProteinsABSTRACT
The study was undertaken to measure plasma interleukin-1 (IL-1) receptor antagonist and IL-10 concentrations in patients with acute myocardial infarction and to analyze their relationship to the hemodynamics, severity, and prognosis of myocardial infarction in its acute stages. We attempted to define the kinetics of IL-1 receptor antagonist and IL-10 in patients with acute myocardial infarction (n = 34, age 42-91 years, mean 68 years). Plasma IL-1 receptor antagonist and IL-10 levels were measured by enzyme-linked immunosorbent assay. Patients in group A (n = 17) had uncomplicated acute myocardial infarction (Killip class I). Patients in group B (n = 17) had severe acute myocardial infarction (Killip class II, III, or IV). Peak Il-1 receptor antagonist and IL-10 levels in group B were significantly higher (p < 0.05) than those of group A. In group B, the peak IL-1 receptor antagonist levels were significantly correlated with white blood cell counts (r = 0.63, p = 0.006), pulmonary capillary wedge pressure (r = 0.78, p = 0.0002), and cardiac index (r = -0.51, p = 0.04). Peak IL-10 levels were significantly correlated with white blood cell counts (r = 0.60, p = 0.01), the pulmonary wedge pressure (r = 0.73, p = 0.0008), and cardiac index (r = -0.50, p = 0.04). Moreover, a significant correlation was found between the peak IL-1 receptor antagonist and IL-10 levels (r = 0.91, p < 0.0001). The peak IL-1 receptor antagonist levels in nonsurvivors (n = 13) were significantly higher (p < 0.01) than those in survivors (n = 21). The plasma IL-1 receptor antagonist and IL-10 levels were closely correlated with the severity of hemodynamics in acute myocardial infarction and with the clinical status of patients with severe acute myocardial infarction. Results suggest that plasma IL-1 receptor antagonist and IL-10 can serve as prognostic indicators in cases of sever acute myocardial infarction.
Subject(s)
Hemodynamics/physiology , Interleukin-10/blood , Myocardial Infarction/blood , Receptors, Interleukin-1/antagonists & inhibitors , Sialoglycoproteins/pharmacology , Adult , Aged , Aged, 80 and over , Female , Humans , Interleukin 1 Receptor Antagonist Protein , Linear Models , Male , Middle Aged , Myocardial Infarction/mortality , Myocardial Infarction/physiopathology , Prognosis , Recombinant Proteins/pharmacologyABSTRACT
We have isolated a new MT-MMP related gene of 3.3 kb from a mouse lung cDNA library using a human MT1-MMP cDNA as a probe. The deduced protein sequence shows 87% homology to human MT2-MMP and 52, 50 and 29% to MT1-MMP, MT3-MMP and MT4-MMP, respectively. Thus the gene is thought to be a mouse homologue of human MT2-MMP. A monoclonal antibody raised against a synthetic peptide recognized mouse MT2-MMP as a 70 kDa protein. Like MT1- and MT3-MMPs, mouse MT2-MMP caused activation of progelatinase A upon co-transfection into COS-1 cells.