الملخص
Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.
الموضوعات
Animals , Female , Mice , Enzyme Inhibitors/pharmacology , Fibrin Tissue Adhesive/pharmacology , Invertebrate Hormones/analysis , Leukocyte Elastase/antagonists & inhibitors , Macrophages/immunology , Skin/drug effects , Tongue/drug effects , Wound Healing/drug effectsالملخص
The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50)= 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.
الموضوعات
Animals , Cattle , Cricetinae , Humans , Biosensing Techniques , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured , Chickens , Endothelial Cells/cytology , Kinetics , Kringles , Ligands , Peptide Fragments/chemistry , Protein Binding , Receptors, Cell Surface/metabolism , Receptors, Urokinase Plasminogen Activator , Urokinase-Type Plasminogen Activator/chemistry , Vascular Endothelial Growth Factor A/pharmacologyالملخص
Deoxyhypusine is a modified lysine and formed posttranslationally to be the eukaryotic initiation factor eIF5A by deoxyhypusine synthase, employing spermidine as butylamine donor. Subsequent hydroxylation of this deoxyhypusine-containing intermediate completes the maturation of eIF5A. The previous report showed that deoxyhypusine synthase was phosphorylated by PKC in vivo and the association of deoxyhypusine synthase with PKC in CHO cells was PMA-, and Ca(2+)/phospholipid-dependent. We have extended study on the phosphorylation of deoxyhypusine synthase by protein kinase CK2 in order to define its role on the regulation of eIF5A in the cell. The results showed that deoxyhypusine synthase was phosphorylated by CK2 in vivo as well as in vitro. Endogenous CK2 in HeLa cells and the cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced or decreased by the addition of CK2 effectors such as polylysine, heparin, and poly(Glu, Tyr) 4:1. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mainly phosphorylated on threonine residue and less intensely on serine. These results suggest that phosphorylation of deoxyhypusine synthase is CK2-dependent cellular event as well as PKC-mediated effect. However, there were no observable changes in enzyme activity between the phosphorylated and unphosphorylated forms of deoxyhypusine synthase. Taken together, besides its established function in hypusine modification involving eIF5A substrate, deoxyhypusine synthase and its phosphorylation modification may have other independent cellular functions because of versatile roles of deoxyhypusine synthase.
الموضوعات
Animals , Cricetinae , Humans , Mice , Casein Kinase II , Cell Line , HeLa Cells , Oxidoreductases Acting on CH-NH Group Donors/genetics , Phosphoamino Acids/metabolism , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/geneticsالملخص
Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca2+, suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca2+/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca2+ plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca2+/phospholipiddependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.
الموضوعات
Animals , Chick Embryo , Female , Mice , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Cell Line , Cricetinae , Phosphorylation , Protein Binding , Protein Kinase C/antagonists & inhibitorsالملخص
Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (rFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these rFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated rFXIII-A were characterized. rFXIII-A (V34L) and rFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type rFXIII-A and V35L variant. However, the activated rFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2- plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.
الموضوعات
Humans , Blood Coagulation Tests , Catalysis , Enzyme Activation/genetics , Escherichia coli/genetics , Factor XIII/genetics , Fibrin/metabolism , Immunoblotting , Leucine/genetics , Mutagenesis, Site-Directed , Polymorphism, Genetic , Recombinant Proteins/genetics , Thrombin/metabolism , Valine/geneticsالملخص
Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.
الموضوعات
Female , Humans , Mice , Adjuvants, Immunologic/administration & dosage , Administration, Intranasal , Animals , Bacterial Toxins/immunology , Bacterial Toxins/genetics , Bacterial Toxins/administration & dosage , Enterotoxins/immunology , Enterotoxins/genetics , Enterotoxins/administration & dosage , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Feces , Gastric Mucosa/microbiology , Gastric Mucosa/immunology , Helicobacter pylori , Immunoglobulin A, Secretory/immunology , Immunoglobulin G/immunology , Mice, Inbred BALB C , Mutagenesis, Site-Directed , ADP Ribose Transferases/immunology , ADP Ribose Transferases/genetics , Nasal Mucosa/immunology , Point Mutation , Urease/immunology , Urease/administration & dosage , Vaccinationالملخص
The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions. Copyright 2000 Academic Press.
الموضوعات
Humans , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Oxidoreductases Acting on CH-NH Group Donors/genetics , Amino Acid Motifs , Amino Acid Sequence , Escherichia coli/genetics , Fungal Proteins/metabolism , Fungal Proteins/genetics , Molecular Sequence Data , NAD/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Kinase C/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Sequence Deletion , Sequence Homology, Amino Acid , Threonine/metabolism , Yeasts/enzymologyالملخص
Angiostatin is a potent angiogenesis inhibitor that is composed of the first four kringles of plasminogen fragment. Angiostatin with one less kringle molecule (kringle 1 to 3) was recently demonstrated to be an effective angiogenic inhibitor. To determine whether recombinant plasminogen kringle 1-3 (rPK1-3) can inhibit the corneal neovascularization induced by potent angiogenic factors; angiogenin, bFGF, or VEGF, hydron polymer discs each containing 2.0 microg of angiogenin, 500 ng of bFGF, or 500 ng of VEGF respectively were implanted into the corneal stroma of 138 rabbit eyes, and then discs each containing 10 microg, 12.5 microg, 20 microg or 30 microg of rPK1-3 were implanted randomly. Discs containing phosphate buffered saline were also implanted as a control. The angiogenesis score on number and length of newly formed vessels on the each of the rabbit's cornea were recorded daily by two observers (blinded). The treated corneas were also examined histologically. Recombinant PK1-3 treated corneas showed less neovascularization induced by all angiogenic factors (p < 0.05). and the extent of inhibition of neovascularization was proportional to the concentration of rPK1-3 (p < 0.05). Histologic examination showed leukocyte infiltration into the corneal stroma on the PBS treated eyes whereas rPK1-3 treated eyes showed only traces of leukocytes. These results of the effective rPK1-3 inhibition of corneal neovascularization induced by angiogenin, bFGF, or VEGF suggest that this angiostatin related fragment, rPK1-3, may be useful in the treatment of various neovascular diseases. Copyright 2000 Academic Press.
الموضوعات
Chick Embryo , Rabbits , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/genetics , Animals , Chorion/drug effects , Chorion/blood supply , Cornea/pathology , Cornea/drug effects , Cornea/blood supply , Endothelial Growth Factors/pharmacology , Fibroblast Growth Factor 2/pharmacology , Kringles/genetics , Lymphokines/pharmacology , Microscopy/methods , Neovascularization, Pathologic/drug therapy , Plasminogen/pharmacology , Plasminogen/genetics , Recombinant Proteins/pharmacology , Recombinant Proteins/genetics , Ribonuclease, Pancreatic/pharmacologyالملخص
Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.
الموضوعات
Cattle , Chick Embryo , Mice , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/isolation & purification , Angiogenesis Inhibitors/genetics , Animals , Blotting, Western , Cell Movement/drug effects , Chorion/pathology , Chorion/drug effects , Circular Dichroism , Collagen/pharmacology , Collagen/isolation & purification , Collagen/genetics , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/drug effects , Endothelium, Vascular/cytology , Escherichia coli/genetics , Fibroblast Growth Factor 2/pharmacology , Neovascularization, Physiologic/drug effects , Peptide Fragments/pharmacology , Peptide Fragments/isolation & purification , Peptide Fragments/genetics , Protein Folding , Recombinant Proteins/pharmacology , Recombinant Proteins/isolation & purification , Recombinant Proteins/genetics , Solubility , Yeasts/geneticsالملخص
Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.
الموضوعات
Female , Mice , Amino Acid Substitution , Animals , Bacterial Toxins/toxicity , Bacterial Toxins/metabolism , Bacterial Toxins/immunology , Bacterial Toxins/genetics , CHO Cells , Cyclic AMP/metabolism , Enterotoxins/toxicity , Enterotoxins/metabolism , Enterotoxins/immunology , Enterotoxins/genetics , Enzyme-Linked Immunosorbent Assay , Escherichia coli/metabolism , Escherichia coli/genetics , Cricetinae , Immunoglobulin A, Secretory/blood , Ileum/metabolism , Immunity, Mucosal , Mice, Inbred BALB C , Mutagenesis, Site-Directed , ADP Ribose Transferases/metabolism , Recombinant Proteins/toxicity , Recombinant Proteins/metabolism , Recombinant Proteins/immunology , Recombinant Proteins/chemistryالملخص
Hepatitis C virus (HCV) E2 protein is known to be one of putative envelope proteins. To develop a sensitive detection method for HCV infected tissues and cells, monoclonal antibodys (MAbs) to the E2 protein of HCV were prepared from mice immunized with recombinant baculovirus-expressing E2 protein (Bac-E2). Several hybridoma clones secreting various levels of MAb were isolated and isotypes of these MAb were determined. One clone (L.2.3.3) was used for ascites production and the E2-MAb was purified and characterized. The L.2.3.3 reacted well with both Bac-E2 and E. coli expressed glutathione-5-transferase-E2 (GST-E2) fusion proteins. Using HCV patient sera, E2 envelope protein was found to be localized in the cell membrane boundary both in CHO cells and insect cells which express HCV E2 protein. Similar result was obtained when same cells were treated with the MAb L.2.3.3. These results demonstrated that Bac-E2 protein is capable of eliciting high titer antibody production in mice.
الموضوعات
Animals , Cricetinae , Humans , Mice , Antibody Formation , Ascites , Cell Membrane , CHO Cells , Clone Cells , Hepacivirus , Hepatitis C , Hepatitis , Hybridomas , Insectaالملخص
The clinical and pathological features of solitary fibrofolliculoma are presented. Solitary fibrofolliculoma is very rarely encountered and to our knowledge, only 7 cases have been reported in the Western literature and no cases have been published in Korea. We experienced a case of solitary fibrofolliculoma occurring in a 56-year-old female, who had a 1.0 cm-sized and slowly growing nodule on her chin. A brief review of the literature, was made especially in relation to the pathological findings and histogenesis of solitary fibrofolliculoma.
الموضوعات
Female , Humansالملخص
Sebaceous trichofolliculoma is a variant of trichofolliculoma which occurs in the sebaceous areas rich in follicles and is a relatively rare skin tumor. This tumor is a clinically and histologically easy tumor to recognize that is well differentiated. We examined a case of a consists of a 21-year-old female who had a pedunculated nodule on her scalp. Microscopically, the tumor was large, had a centrally located cavity lined by squamous epithelium and radially arranged sebaceous follicles connected to the cavity. No cytological atypia or recurrence after excision was found.
الموضوعات
Female , Humansالملخص
Proliferating trichilemmal tumor is relatively rare, and is generally considered to be a benign tumor that can be histologically mistaken for well-differentiated squamous cell carcinoma. The proliferating trichilemmal tumor is thought to be a tumor with differentiation toward the hair structure because the characteristic trichilemmal keratinization in this tumor is analogous to that of the outer root sheath of anagen hair or the trichilemmal sac surrounding catagen hair. We report four cases of proliferating trichilemmal tumor removed by surgical excision.
الملخص
This clinical and histopathological study was performed with 147 cases of benign mielanocytic tumors and 19 cases of malignant melanomas, which were obtained as surgical specimens from 1974 to 1984 at Department of Clinical Pathology, Catholic Medical College. The results were as follows: 1, In 89 cases of acquired benign melanocytic nevi, the average age of intradermal type(64 cases) was 34. 7 years and that of compound type(24 cases) was 24. 6 years. 2. In 30 cases of congenital nevus, nevus cells were present in the lower two thirds of reticular layer of the dermis in 93. 3% and in the subcutis as well in 3.3%. A case of giant congenital nevus with balloon cell appearance was found. 3. Of the 147 benign melanocytic tumors, a pigmented spindle cell nevus and a desmoplastic nevus were observed. 4. Blue nevi were 11 in number and excised from the face in 7, buttock in 2, shoulder in 1, upper arm in 1, and all were common type histopathologically. 5. Twelve malignant melanornas which were possible to be re-examined histopathologically were composed of 5 nodular type, 3 acral lentiginous type, 1 superficial spreading type and 3 metastatic malignant melanoma.