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PubMed2003

A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps.

Endochondral bone formation has been fairly well characterized from a morphological perspective and yet this process remains largely undefined at molecular and biochemical levels. In vitro and in vivo studies have shown that human bone morphogenetic protein-2 (hBMP-2) is an important developmental growth and differentiation factor, capable of inducing ectopic bone formation in vivo. This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1), runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1) and X (Col10a1). In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1) and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation. In conclusion, oligonucleotide arrays enabled a broader view of endochondral bone formation than has been reported to date. An increased understanding of the roles played by these gene products will improve our understanding of skeletogenesis, fracture repair, and pathological conditions such as osteoporosis.

Animals↗

PubMed2001

Stimulation of RecA-mediated D-loop formation by oligonucleotide-directed triple-helix formation: guided homologous recombination (GOREC).

Oligonucleotide-directed triple helix formation provides an elegant rational basis for gene-specific DNA targeting and has been widely used to interfere with gene expression ("antigene" strategies) and as a molecular tool for biological studies. Various strategies have been developed to introduce sequence modifications in genomes. However, the low efficiency of the overall process in eucaryotic cells impairs efficient recovery of recombinant genomes. Since one limiting step in homologous recombination is the targeting to the homologous sequence, we have tested the contribution of an oligonucleotide-directed triple helix formation on the RecA-dependent association of an oligonucleotide and its homologous target on duplex DNA (D-loop formation). For this study, the recombinant ssDNA fragment was noncovalently associated to a triple helix-forming oligonucleotide. The physicochemical and biochemical characteristics of the triple helix and D-loop structures formed by the complex molecules in the presence or in the absence of RecA protein were determined. We have demonstrated that the triple helix-forming oligonucleotide increases the efficiency of D-loop formation and the RecA protein speeds up also the triple helix formation. The so-called "GOREC" (for guided homologous recombination) approach can be developed as a novel tool to improve the efficiency of directed mutagenesis and gene alteration in living organisms.

Base Sequence↗

PubMed1994

Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation.

Osteopontin is a phosphorylated sialoprotein containing a conserved sequence of contiguous aspartic acid residues. This protein is expressed at high levels in mineralized tissues and has previously been shown to inhibit the in vitro formation of hydroxyapatite (HA). In the present study, protein modification and model compound studies have been used to identify the structural features of osteopontin that are responsible for its crystal-modulating properties. Using metastable calcium phosphate solutions buffered by autotitration, osteopontin caused half-maximal inhibition of HA formation at a concentration (IC50) of 0.06 microgram/ml. The hen egg yolk phosphoprotein phosvitin was a much weaker inhibitor, while dextran sulphate had no effect. The synthetic polypeptide poly(aspartic acid) was almost as effective an inhibitor of HA formation as osteopontin (IC50 0.11 microgram/ml), whereas poly(glutamic acid) was more than a thousand times less potent (IC50 155 micrograms/ml). In a steady-state agarose gel system, much higher polypeptide concentrations were required for inhibition of HA formation, but a similar relative order of inhibitory effectiveness was observed. Treatment of osteopontin with alkaline phosphatase removed 84% of the covalently bound phosphate and reduced its HA-inhibiting activity by more than 40-fold. Treatment with glycine ethyl ester in the presence of carbodi-imide modified 86% of the carboxylate groups in osteopontin and reduced its inhibitory activity by 6-fold. These findings indicate that osteopontin is a potent inhibitor of HA formation. This activity requires phosphate and carboxylate groups, possibly including the conserved sequence of contiguous aspartic acid residues. Osteopontin may act as an inhibitor of phase separation in physiological fluids of high supersaturation.

Amino Acid Sequence↗

PubMed2000

The effect of alpha-phenyl-tert-butyl nitrone (PBN) on free radical formation in transient focal ischaemia measured by microdialysis and 3,4-dihydroxybenzoate formation.

alpha-phenyl-tert-butyl nitrone (PBN) reduces infarct size, improves recovery of brain energy metabolism and delays the secondary increase in extracellular potassium after focal ischaemia, presumably by trapping OH radicals. We investigated the effect of PBN on the formation of 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of OH radical formation, during and following middle cerebral artery occlusion (MCAO). Rats, subjected to 2 h of ischaemia followed by 3 h of recirculation, were injected with either vehicle or PBN (100 mg kg-1 i.p.) prior to MCAO or immediately after recirculation, respectively. The in vivo microdialysis technique was used to collect samples for analysis of 3,4-DHBA by HPLC. The basal levels of 3,4-DHBA were 56-77 nmol L-1 in the four groups. During ischaemia, the formation of 3,4-DHBA decreased by about 50% in all groups. Upon recirculation, a 3-fold rise in 3,4-DHBA formation was seen. At 2 h of recirculation the mean value of 3,4-DHBA in the pretreated, vehicle-injected animals was 125 +/- 18 nmol L-1 and in the PBN-injected 145 +/- 48 nmol L-1, respectively. When the animals were treated after MCAO either with vehicle or PBN the values at 2 h recirculation were 155 +/- 148 and 189 +/- 145 nmol L-1, respectively. No statistically significant difference between vehicle- and PBN-injected groups was seen. We conclude that during reperfusion following MCAO, hydroxyl radical formation increases. The increase is not ameliorated by PBN which suggests that PBN does not protect the brain by a general scavenging of OH radicals, although tissue specific actions cannot be excluded.

Animals↗

PubMedDate not supplied

Effects of transforming growth factor beta1 on bonelike tissue formation in three-dimensional cell culture. I. Culture conditions and tissue formation.

Bone tissue engineering based on growing bone marrow stromal cells on poly(L-lactic-co-glycolic acid) fiber meshes suffers from limited matrix production and mineralization when the cells are cultured with the standard differentiation supplements (dexamethasone, beta-glycerophosphate, and ascorbic acid). To overcome this problem we included transforming growth factor beta1 (TGF-beta1), which is described as playing a key role in collagen type I formation, although its effect on mineralization is controversially discussed. The investigations focused on establishing culture conditions for the application of TGF-beta1 in three-dimensional cell culture and on the effects of different doses of TGF-beta1 (1-20 ng/mL) on bonelike extracellular matrix formation. Immunohistochemical staining showed that TGF-beta1 enhanced the formation of procollagen type I, collagen type I, and collagen type V, especially under dynamic culture conditions (orbital shaker). A long-term study confirmed positive effects on the formation of extracellular matrix, which penetrated the scaffold to a depth of 250 to 300 microm. Mineralization, qualified by scanning electron microscopy in combination with energy-dispersive X-ray analysis and evaluated by determination of the Ca2+ content per scaffold, was up to 1.7-fold increased by TGF-beta1 compared with the control. In conclusion, the growth factor TGF-beta1 seems to be effective in improving extracellular bonelike matrix formation in vitro.

Animals↗

PubMed1995

Early formation of DNA adducts compared with tumor formation in a long-term tumor study in rats after administration of 2-nitrofluorene.

2-Nitrofluorene (NF) is a model compound for nitroarenes which has been identified in diesel exhaust and in urban air. The current study was carried out to observe the carcinogenicity of different doses of NF to rats and DNA adduct formation in different organs at an early stage of NF administration. One group of rats was fed basal diet as a control, whereas the other three groups of rats were fed basal diet supplemented with different amounts of NF (0.24, 0.95 and 2.37 mmol NF/kg diet, referred to as low, medium and high dose, respectively). The rats were exposed to NF continuously for 11 months, after which all groups of rats were fed basal diet without NF for another 13 months. In the high dose group hepatocellular carcinomas were found in all rats (20/20), forestomach squamous carcinomas in 11 and cortical kidney carcinomas in 10 rats. Fifteen out of 19 rats fed the medium dose of NF had hepatocellular carcinomas, 16 had forestomach squamous carcinomas and 15 had cortical kidney carcinomas. The major tumors of the rats fed the low dose of NF were forestomach squamous carcinomas (10/18). DNA adducts formed in tumor target organs after 1, 2, 6 and 10 days NF administration were dose- and time-dependent. Ten days after the start of NF administration DNA adduct levels were found to be 54, 11 and 6 DNA adducts/10(8) normal nucleotides in forestomach, liver and kidney respectively. In the non-tumor target organs levels in the range 1.7-4.8 DNA adducts/10(8) normal nucleotides were found. DNA adduct formation in this study showed a good correlation with the localization of tumors, although there is a need for additional factors for tumor formation. The results indicate that DNA adduct formation is an important factor for tumor formation and suggest that DNA adducts could be used as biomarkers for genotoxic risk.

Animals↗

PubMed1991

HIV-induced syncytium formation requires the formation of conjugates between virus-infected and uninfected T-cells in vitro.

The transmission of HIV requires the interaction of the cell-surface CD4 receptor and the viral envelope glycoprotein. Experiments were performed to determine the role of other cell-surface molecules in the development of HIV-induced syncytia. Although CEM and MT-2 cells had similar cell-surface CD4 receptor densities, less than 1% of CEM cells and greater than 95% of MT-2 cells formed syncytia with H9 cells chronically infected with HIV-1 (H9-IIIB). When compared with CEM cells, MT-2 cells exhibited a 10-fold and threefold greater capacity to form homotypic and heterotypic conjugates with H9 cells, respectively. Increasing the conjugate formation capacity of CEM cells with the lectin wheat germ agglutinin led to a greater than 30-fold increase in the formation of syncytia with H9/IIIB cells. The formation of syncytia between MT-2 and H9/IIIB cells was magnesium-, energy-, temperature-, and actin-cytoskeleton-dependent, and could be inhibited (65%) by an anti-LFA-1 monoclonal antibody. The combination of anti-leukocyte function-associated antigen-1 (LFA-1) and anti-CD2 monoclonal antibodies resulted in a synergistic inhibition (89%) of syncytium formation. These results indicate that integrins and other cell-surface adhesion molecules regulate HIV-induced syncytium formation.

CD4 Antigens↗

PubMed2001

Incidence and management of esophageal stricture formation, ulcer bleeding, perforation, and massive hematoma formation from sclerotherapy versus band ligation.

OBJECTIVES: The aim of this study was to compare the incidence and endoscopic management of esophageal stricture formation, significant ulcer bleeding, massive esophageal hematoma, and perforation resulting from endoscopic band ligation or sclerotherapy of esophageal varices. METHODS: Consecutive esophagogastroduodenoscopies in which band ligation or sclerotherapy was performed for acute or obliterative therapy were entered into a computerized endoscopy database during a 7-yr period. Patients were excluded if they died within 72 h of treatment session from complications unrelated to the procedure. Sclerotherapy was performed using a 25-gauge needle with 1.5% sodium tetradecyl sulfate and banding was primarily performed with a Wilson-Cook 6 or 10 shooter. Complications were assessed at scheduled endoscopy and outpatient clinic visits, review of quality assurance data tallied on a monthly basis, and patient records. RESULTS: Two hundred twenty-one cases of sclerotherapy were performed in 59 patients compared to 110 cases of band ligation in 52 patients. Five patients were excluded because of death within 72 h of the procedure. The incidence of complications from sclerotherapy:banding on a per patient basis included: esophageal stricture formation 25.6%:1.9%, ulcer bleed 25.4%:5.7%, esophageal perforation 2.2%:0%, and massive esophageal hematoma 1.6%:0%. A significant difference in complications between sclerotherapy and band ligation was noted for both stricture formation (p < 0.0005) and ulcer bleeding (p < 0.0001). The majority of ulcer bleeds required no therapeutic intervention, whereas stricture formation required multiple dilation sessions. CONCLUSIONS: Band ligation has a significantly lower incidence of stricture formation and ulcer bleeding compared to sclerotherapy. The majority of complications can be managed with endoscopic interventions.

Databases, Factual↗

PubMed2001

Evidence of macrophage foam cell formation by very low-density lipoprotein receptor: interferon-gamma inhibition of very low-density lipoprotein receptor expression and foam cell formation in macrophages.

BACKGROUND: Expression of the VLDL receptor, primarily in macrophages, has been confirmed in human and rabbit atherosclerotic lesions. The high binding affinity of the VLDL receptor for remnant particles implicates the VLDL receptor pathway in the foam cell formation mechanism in macrophages. This study investigates the effect of interferon (IFN)-gamma on VLDL receptor expression in phorbol-12-myristate-13-acetate (PMA)-treated THP-1, HL-60 macrophages, and human monocyte-derived macrophages. METHODS AND RESULTS: THP-1 cells were induced to differentiate into macrophages by PMA treatment. IFN-gamma was added to the medium, and expression of the VLDL receptor was determined. (125)I-beta-VLDL degradation study and oil red O staining were examined. In THP-1 macrophages, VLDL receptor protein expression decreased at 2 days after PMA treatment but increased at 3 days and increased up to 5 days. Scavenger receptor proteins, which were not originally present, appeared at 3 days after PMA treatment. IFN-gamma inhibited VLDL receptor expression in a dose-and time-dependent manner in macrophages. However, no inhibitory effect was observed in monocytes. Moreover, IFN-gamma receptor mRNA increased during differentiation to macrophages. (125)I-beta-VLDL degradation study and oil red O staining showed that IFN-gamma significantly inhibited foam cell formation after the uptake of beta-VLDL. LDL receptor-related protein (LRP) and LDL receptor mRNAs were not expressed in macrophages. In PMA-treated HL-60 macrophages and human monocyte-derived macrophages, IFN-gamma also inhibited VLDL receptor expression and foam cell formation by beta-VLDL. CONCLUSIONS: VLDL receptor expression is upregulated during monocyte-macrophage differentiation. IFN-gamma inhibits VLDL receptor expression and foam cell formation only in macrophages. Remnant particles induce macrophage foam cell formation through the VLDL receptor pathway.

Cell Differentiation↗

PubMed1985

Mutual desmosome formation between all binary combinations of human, bovine, canine, avian and amphibian cells: desmosome formation is not tissue- or species-specific.

Our previous work has suggested that the molecular components of desmosomes are highly conserved between different tissues and different vertebrate species. In order to determine whether the adhesion recognition mechanism of desmosomes is also conserved we have examined the specificity of desmosome formation between different epithelial cell types by co-culturing binary combinations of cells from different species and from epidermal and non-epidermal origin. The following cell types were used: human (HeLa, cervical carcinoma), bovine (Madin Darby bovine kidney, MDBK), canine (Madin Darby canine kidney, MDCK), avian (chick embryonic corneal epithelium) and amphibian (Rana pipiens, adult corneal epithelium). Different cells in co-culture were identified on the basis of at least one of the following criteria: (1) morphology by phase-contrast microscopy; (2) presence or absence of staining of cytokeratin with monoclonal antibody LE61; (3) morphology at the electron microscope level. Mutual desmosome formation between different cell types was assessed using fluorescent antibody staining with anti-desmoplakin antibodies and confirmed using electron microscopy. We have found that mutual desmosome formation occurred between all binary combinations of human, bovine, canine, avian and amphibian cells. Thus there is complete non-selectivity of desmosome formation between five different epithelial cell types from three vertebrate classes. Our results suggest that desmosome formation is not tissue- or species-specific and that the mechanism for intercellular binding involved in desmosomal adhesion is highly conserved.

Animals↗

PubMed2005

Adaptive immune responses are dispensable for isolated lymphoid follicle formation: antigen-naive, lymphotoxin-sufficient B lymphocytes drive the formation of mature isolated lymphoid follicles.

Isolated lymphoid follicles (ILFs) are recently appreciated members of the mucosal immune system. The architecture, composition, and inducible nature of these structures indicates that these structures are tertiary lymphoid structures. The process leading to the formation of tertiary lymphoid structures, lymphoid neogenesis, has been observed in a number of inflammatory and autoimmune conditions. Given this association, there is considerable interest in identifying the factors promoting lymphoid neogenesis, and understanding the steps in this process. Using murine ILF formation as a model, we have examined the roles of different cellular sources of lymphotoxin (LT) and the adaptive immune response in lymphoid neogenesis. In this study, we report that, although other cellular sources of LT may supplant B lymphocytes in the formation of immature ILFs (loosely organized clusters of B lymphocytes), LT-sufficient B lymphocytes are required for the progression of immature ILFs to mature ILFs (organized lymphoid aggregates with a follicle-associated epithelium). ILF formation occurs in the absence of T lymphocytes and Ag-specific B lymphocyte responses, and ILF B lymphocytes express elevated levels of LT in the absence of antigenic stimulation. Consistent with a role for chemokines inducing LT expression in Ag-naive B lymphocytes, and a chemokine-driven positive-feedback loop driving mature ILF formation, mature ILFs express elevated levels of B lymphocyte chemoattractant in the absence of Ag-specific B lymphocyte stimulation. These observations indicate that ILFs contain Ag-naive lymphocytes, and suggest that events occurring within ILFs shape subsequent immune responses mediated by these lymphocytes.

Animals↗

PubMed1975

[Formation of spontaneous and immune rosettes by the cells of the thymus and other formations of the rabbit lymphoid system].

Only T1--RFC (rosette-forming cells) are revealed in the thymus of nonimmunized rabbits. Their number is 2--2.5 times less than in the palatine tonsils, submaxillary lymph nodes and the spleen. T2--RFC are present in these lymphoid formations. There is an increase in the T1--RFC in the thymus after the intravenous immunization of rabbits with sheep erythrocytes. In other lymphoid formations the correlation of the population of cells of the thymus origin altered as a result of increase in the number of T2--RFC. B--RFC accumulated in considerable amounts. Dynamics of T2 and B--RFC accumulation in the lymphoid formations corresponded to the highest antibody titres in the rabbit blood. In the formation of primary immune response the amount of the T1 and T2-RFC in the formations of rabbit lymphoid system depended on the dose of the antigen.

Animals↗

PubMed1982

Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation.

Protein C is activated rapidly when thrombin binds to a specific cell surface cofactor protein, thrombomodulin. Studies were initiated to determine the influence of thrombin-thrombomodulin complex formation on the substrate specificity of thrombin. When thrombin binds to thrombomodulin, the resultant complex retains less than 1% of the fibrinogen clotting activity of free thrombin. Permanent alteration of the thrombin molecule is not involved since full clotting activity is regenerated by incubation of the complex with excess diisopropyl phosphothrombin. Unlike the activation of protein C by the thrombin-thrombomodulin complex which is dependent on Ca2+, inhibition of fibrinogen clotting activity is not dependent on the presence of divalent metal ions. Formation of the thrombin-thrombomodulin complex also inhibits thrombin activation of factor V. Despite these changes in macromolecular substate specificity, no significant change in the hydrolysis of the synthetic substrates p-tosyl-L-arginine methyl ester and N alpha-benzoyl-L-arginine ethyl ester is detected upon formation of the thrombin-thrombomodulin complex. Formation of this complex results in a slight increase in the Km (from 9.0 +/- 0.4 to 10.2 +/- 0.6 microM) and Vmax (from 230 +/- 10 to 270 +/- 10 mol/s/mol of thrombin) for the specific thrombin substrate H-D-Phe-Pip-Arg-p-nitroanilide. These studies suggest that thrombomodulin has two distinct anticoagulant functions: 1) to inhibit the ability of thrombin to clot fibrinogen and to activate factor V; and 2) to accelerate the formation of the anticoagulant, activated protein C.

Animals↗

PubMed1982

Formation of IgE-binding factors by rat T lymphocytes. VI. Cellular mechanisms for the formation of IgE-potentiating factor and IgE-suppressive factor by antigenic stimulation of antigen-primed spleen cells.

Analysis of cellular mechanisms of formation of IgE-binding factors by KLH-primed spleen cells revealed that the presentation of KLH to KLH-primed T cells by adherent cells resulted in the formation of lymphokines that in turn stimulated unprimed lymphocytes to form IgE-binding factors. Lymphokines released from KLH-alum-primed spleen cells induced normal lymphocytes to form IgE-potentiating factors, whereas those released from KLH-CFA-primed spleen cells induced the formation of IgE-suppressive factors. Fractionation of the lymphokines from KLH-primed spleen cells and analysis of cell sources of the lymphokines revealed that multiple factors are involved in the selective formation of one or the other IgE-binding factors. Thus, KLH-alum primed splenic T cells from "inducers" of IgE-binding factors and glycosylation-enhancing factors upon antigenic stimulation, and these factors in combination stimulate unprimed W 3/25+ Fc gamma R+ T cells to form IgE-potentiating factors. Antigenic stimulation of KLH-CFA-primed T cells results in the formation of the "inducers" and glycosylation-inhibiting factors, and these two lymphokines collectively stimulate unprimed W 3/25+ Fc gamma R+ T cells to form IgE-suppressive factors. Cell sources of "inducers" are W 3/25+ Fc gamma R- T cells, cells different from the source of IgE-binding factors. The glycosylation-enhancing factor is derived from W 3/25+ F gamma R+ T cells in KLH-alum-primed spleen; the glycosylation-inhibiting factor is derived from OX 8+ T cells in KLH-CFA-primed spleen. Evidence was obtained that these lymphokines, which modulate the protein glycosylation of IgE-binding factors during their biosynthesis, are derived from antigen-primed T cells and determine the nature of IgE-binding factors formed.

Animals↗

PubMed1980

[Genetic control of plasmid F' formation. II. Role of recipient recA- in the process of plasmid F' formation].

We have chosen the process of F'-plasmid formation as a model for a study of the illegitimate, recA-independent recombination. Our genetic data showed that F'-formation was under genetic control of the Hfr donor cells. To determine the role of recipient cells in the process of F-prime formation we compared the pattern of transconjugant formation in the crosses of Hfr and F' donors with the recA-nalA+ and recA-nalA- recipients, nalA- mutation in the recipient lead to the sharp decrease of the total yield of transconjugants selected for proximal markers and decrease the fraction of conjugative plasmids in progeny. The plasmids inherited in the nalA- recipient in HfrxF- crosses possess only proximal selected markers while the appropriate distribution of proximal and more distal markers took place in the nalA+ recipient. The homogeneity for the sensitivity of nalA- progeny to the male specific phages in comparison with the nalA+ transconjugants was also observed. The data obtained show that the nalA- mutation has a strong influence on the process of final inheritance and probably formation of F'-plasmids in the recipient cells. The extent of the influence of the nalA- mutation on the pattern of F'-plasmid inheritance was dependent upon the primary F' structure formed (or existed) in the donor cell.

Conjugation, Genetic↗

PubMedDate not supplied

[Effect of the length of molecular chain on the formation of triple-helical collagen-like complex in solutions. The role of water in the formation of triple helix].

Effect of molecular chain length on the formation of the collagen-like triple helix in synthetic oligotripeptides Z-(Gly-Pro-Pro)n-OMe (n-1,2,3,...,8) in solutions has been studied, using IR- and CD-spectroscopy methods. The helix formation under different conditions is investigated: in the presence of a relatively inert solvent (chloroform), in the presence of a hydrogen bond acceptor (dioxan), in the presence of a hydrogen bond acceptor and donor as well (ethanol). Special attention is paid to the role of water in the formation of a stable triple-helical structure. Successive stages in the formation of a stable triple-helical structure in solutions during elongation of the peptide chain is revealed, which may be correlated with the helix nucleation process. A minimum peptide length, when peptide chains are still able to associate in the collagen-like triple-helical complex, involves three triplets for oligomers in chloroform solution, four triplets for oligomers in dioxan and ethanol solutions, six triplets for oligomers in aqueous solution. The main features of the completed triple-helical structure in water are found already in the oligotripeptide with n-8, where the formation one full turn of the superhelix is finished.

Circular Dichroism↗

PubMed1998

Fibrin-rich and platelet-rich thrombus formation on neointima: recombinant tissue factor pathway inhibitor prevents fibrin formation and neointimal development following repeated balloon injury of rabbit aorta.

Thrombus formation and neointimal growth are the critical events in restenosis after balloon angioplasty. However, the responses of diseased vessels to injuries caused by balloon angioplasty have not been well examined. We investigated the thrombus formation and neointimal development following the balloon injury to the previously induced neointima in the rabbit aorta and the effects of recombinant tissue factor pathway inhibitor (rTFPI) on these responses. Rabbit thoracic aortas were subjected to injury with a Fogarty 4F balloon catheter at 1.75 atm (first injury), and 4 weeks later the same vessels were subjected to the second injury with a Swan-Ganz 5F balloon catheter at 1.4 atm (mild-injury group) or 1.8 atm (severe-injury group), and immediately after that a retrograde bolus injection of rTFPI (100 microg/kg body weight) or saline was performed into the injured segments via the central tube of the Swan-Ganz catheter. Twenty minutes after the second injury, the injured surfaces were covered with platelet-rich thrombi in the mild-injury group and with fibrin-rich thrombi in the severe-injury group. Damaged intimal smooth muscle cells, which were immunohistochemically positive for tissue factor (TF), were observed beneath the fibrin-rich thrombi. The neointima 4 weeks after the second injury was significantly thicker in the severe-injury group than in the mild-injury group. The bolus infusion of rTFPI markedly inhibited fibrin formation on the injured surfaces, and significantly reduced the neointimal development in the severe-injury group at 4 weeks after the second injury. These results indicate that TF-dependent coagulation pathway is primarily responsible for fibrin-rich thrombus formation and may play an important role in neointimal development following the balloon injury to the rabbit aortic neointima. Additionally the bolus administration of rTFPI to the injured vessels could prevent mural thrombus formation and neointimal growth after balloon angioplasty.

Animals↗

PubMed1963

Antibody formation. IV. Formation of rapidly and slowly sedimenting antibodies and immunological memory to bacteriophage phi-X 174.

Injection of a sufficient dose of bacteriophage phiX 174 into guinea pigs results in the formation of rapidly sedimenting antibody molecules (19S), and later, slowly sedimenting molecules (7S). Above a threshold dose of antigen, the relative rate of 19S formation is maximal and dose-independent; below this dose, slower relative rates are obtained. The time for doubling the serum 19S level is as short as 6 to 8 hours, suggesting that the absolute rate of antibody formation per cell is increasing in addition to proliferation of antibody-producing cells. Synthesis of 19S after injection of 10(10) phiX virtually ceases at 10 days after which 19S antibody activity disappears from the circulation with a half-life of approximately 24 hours. A second injection of phiX on day 5 or 9 prolongs 19S synthesis, indicating that antigen not only can regulate the relative rate, but also is essential for continued synthesis of 19S. 19S synthesis is also prolonged in guinea pigs by injection of phiX with endotoxin or by 400 r whole body x-irradiation 24 hours after injection of phage into rabbits. The primary 7S response is not detected until approximately 1 week after immunization and relative rates are antigen-dependent. Primary 7S synthesis can continue for many months and leads to preparation for a secondary antibody response (immunological memory) during which only 7S is detected. In contrast, in animals that form precipitating 19S without detectable 7S, a second injection of phage 1 month later results in a second 19S response which closely resembles the first. These findings have led to the suggestion that formation of 19S does not lead to persisting immunological memory.

Animals↗