Showing papers on "Proteolytic enzymes published in 1986"
TL;DR: The data confirmed that antimicrobial activity of L. helveticus 481 was mediated by a bacteriocin, designated helveicin J, and provided evidence for a chromosomal location of helveticsin J and host immunity determinants.
Abstract: Lactobacillus helveticus 481 produced an antimicrobial agent active against five closely related species. The sensitive indicators included L. helveticus 1846 and 1244, L. bulgaricus 1373 and 1489, and L. lactis 970. The antimicrobial compound was active at neutral pH under aerobic or anaerobic conditions, was sensitive to proteolytic enzymes and heat (30 min at 100 degrees C), and demonstrated a bactericidal mode of action against sensitive indicators. These data confirmed that antimicrobial activity of L. helveticus 481 was mediated by a bacteriocin, designated helveticin J. Production of helveticin J was maximized in an anaerobic fermentor held at a constant pH of 5.5. Ultrafiltration experiments on culture supernatants containing the bacteriocin revealed that helveticin J was present as an aggregate with a molecular weight in excess of 300,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of helveticin J purified through Sephadex chromatography resolved a 37,000-dalton protein band with bacteriocin activity. L. helveticus 481 was shown to harbor a single 8-megadalton plasmid (pMJ1008). Isolates cured of pMJ1008 were phenotypically identical to plasmid-bearing cells in fermentation patterns, helveticin J activity, and immunity spectra. The data provided evidence for a chromosomal location of helveticin J and host immunity determinants.
405 citations
TL;DR: Evidence is presented to suggest that proteolytic systems (of proteinases, proteases, and peptidases) may function to prevent the formation or accumulation of oxidatively damaged protein aggregates and to prevent a wide variety of potentially toxic consequences.
293 citations
TL;DR: The results obtained strongly suggest that A-CAM is a Ca2+-dependent adherens junction- specific membrane glycoprotein that is involved in intercellular adhesion in these sites.
Abstract: The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane-bound plaques of the fascia adherens. In cultured chick lens cells, A-CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A-binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451-1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction-specific membrane glycoprotein that is involved in intercellular adhesion in these sites.
250 citations
TL;DR: It is concluded that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative.
Abstract: The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.
224 citations
TL;DR: A cDNA clone from a human liver library which codes for HI-30 is isolated, and its DNA sequence is determined, which not only codes forHI-30 but also another serum protein, alpha-1-microglobulin, which has not been previously associated with ITI or HI- 30.
Abstract: Inter-alpha-trypsin inhibitor (ITI) is a 180 kd serine proteinase inhibitor found in human serum. Treatment of 180 kd ITI with trypsin releases a 30 kd fragment (HI-30) which contains the anti-proteolytic activity of the high molecular weight form. We have isolated a cDNA clone from a human liver library which codes for HI-30, and have determined its DNA sequence. The mRNA not only codes for HI-30 but also another serum protein, alpha-1-microglobulin, which has not been previously associated with ITI or HI-30. The alpha-1-microglobulin sequence is found in the amino-terminus of the protein and is preceded by a signal sequence. HI-30 is found at the carboxy-terminus. The two protein sequences are separated by two arginine residues.
201 citations
TL;DR: Data support the validity of the "protease- Protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysematous lung tissue.
Abstract: The current working hypothesis concerning the pathogenesis of human pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes into the interstitium. These enzymes, in particular elastase, can bind to and degrade interstitial elastin. This report describes an immunohistochemical, ultrastructural technique that utilizes polyclonal antibodies to localize neutrophil elastase in human lungs. Using both the immunoperoxidase and the immunogold methods on thin, embedded sections of surgically resected human emphysematous lung tissue, elastase was localized in neutrophils in the lung interstitium and extracellularly in association with interstitial elastic fibers in human lungs that showed local emphysema of varying severity. Quantitative morphometric data were obtained from the lungs of eight patients undergoing lobectomy for removal of pulmonary carcinomas. Patients had preoperative forced expiratory volume (FEV1)% levels ranging from 55 to 77. There was a correlation between a quantitative measure of the local distribution of neutrophil elastase in contact with alveolar interstitial elastin and the local presence of emphysematous change as determined by mean linear intercept of the various histologic sections. These data support the validity of the "protease-protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysema.
190 citations
TL;DR: These findings imply that the release of either fibrinopeptide triggers similar modes of aggregation; the intermolecular binding sites can be localized to particular molecular domains.
189 citations
TL;DR: These modified enzymes are examples on how the different activities of an enzyme can be perturbed by “protein engineering”, hence rendering the enzyme particularly suitable for certain processes.
Abstract: Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases. Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many fungi, in addition, excrete serine carboxypeptidases. Apparently, bacteria do not employ this group of enzymes.
174 citations
TL;DR: Injection of nontoxic doses of CCK-8 before proglumide or gabexate injections completely abolished all beneficial effects and also increased the severity of pancreatitis due to CDE diet alone.
Abstract: The effects of the cholecystokinin (CCK)-receptor antagonist proglumide, the protease inhibitor gabexate, and the hormones secretin and cholecystokinin-octapeptide (CCK-8) were studied in a model of acute hemorrhagic pancreatitis induced by feeding mice a choline-deficient, ethionine-supplemented (CDE) diet. Injections of gabexate and proglumide from initiation of CDE diet (before induction of pancreatitis) increased survival from 37% (diet alone) to 85 and 75%, respectively, and also ameliorated histological alterations and increases in serum amylase concentration and pancreatic activated trypsin. Secretin had no major beneficial effect. When proglumide or gabexate were given after induction of pancreatitis, proglumide still increased survival to 75%, whereas gabexate no longer did. Injection of nontoxic doses of CCK-8 before proglumide or gabexate injections completely abolished all beneficial effects and also increased the severity of pancreatitis due to CDE diet alone. Blockade of CCK receptors and early inhibition of protease activity may be beneficial in severe acute pancreatitis. Cholecystokinin appears to play a contributory role in the development of pancreatitis.
169 citations
TL;DR: In the seminiferous epithelium, a Sertoli cell-derived growth factor is suggested to have a role in local regulation together with other factors, such as meiosis-inducing and -preventing substances, a somatomedin-like compound, and the spermatogonial chalone.
Abstract: Publisher Summary This chapter discusses the cell interactions during the seminiferous epithelial cycle. Spermatozoa belong to the most differentiated cells of the body. Their special features are a haploid number of chromosomes, a tightly packed inactive form of the chromatin, a small amount of cytoplasm, and an ability of independent movement by a flagellum. The development of spermatozoa in the seminiferous epithelium includes three main phases: (1) spermatogonial multiplication, (2) meiosis, and (3) spermiogenesis. Cells in these phases are called spermatogonia, spermatocytes, and spermatids, respectively. The stage-dependent variation of the hormone responses in the seminiferous epithelium strongly suggests an existence of local paracrine regulation and cell interaction mechanisms in the seminiferous epithelium, that are dependent on spermatogenic cells associated with the Sertoli cells at each stage of the cycle of the seminiferous epithelium. The nature of this interaction is obscure; however some advances have been made. The secretion of a proteolytic enzyme, urokinase-type plasminogen activator, seems to be dependent on both cellular and hormonal regulation in the seminiferous epithelium. Testicular GnRH-like factors and proopiomelanocortin-derived peptides may play a role in seminiferous tubule-Leydig cell interaction. In the seminiferous epithelium, a Sertoli cell-derived growth factor is suggested to have a role in local regulation together with other factors, such as meiosis-inducing and -preventing substances, a somatomedin-like compound, and the spermatogonial chalone.
158 citations
TL;DR: The results indicate that the heat-induced changes in hemagglutinin which allow fusion activity result in partial denaturation of the molecule, and the changes in structure required for fusion at low pH and normal temperature are specifically restricted.
TL;DR: Analysis of the amino acids in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles suggests that these linkages involve the phosphate centres of the caseins.
Abstract: The strength of binding of the individual caseins and the nature of the bonding within bovine casein micelles were examined through dissociation of the micelles by dialysis of skim milk either against phosphate-free buffers containing 3 or 6 mm-CaCl2, or against buffers that were nearly saturated with respect to micellar calcium phosphate, but which had a free Ca2+ concentration in the range 0·4–5·9 mm. Dissociation was followed by ultracentrifuging the dialysed milks and determining the partition of the total and the individual caseins between the pellet and serum. During dialysis against the phosphate-free buffers both colloidal Ca and Pi in the milks decreased and about 30 % of the Pi could be removed without significant casein dissociation. With further loss of Pi, however, increasing dissociation occurred and the proportions of the individual caseins retained in the casein pellet were in the order αs2- > αs1- > β- ≈ κ-casein. Dialysis against the calcium phosphate buffers resulted in no loss of colloidal Pi but colloidal Ca increased with the free Ca2+ concentration of the buffer. Little change in the casein partition occurred in the presence of more than 1 mm free Ca2+, but serum casein increased markedly at lower levels, and the strength of binding of the individual caseins in the pelleted casein was in the order αs2-> αs1- > β- > κ-casein. In both types of buffer, dissociation is considered to occur through the breaking of linkages between the caseins and inorganic constituents. Analysis of the amino acids in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles suggests that these linkages involve the phosphate centres of the caseins.
TL;DR: In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined, suggesting a probable molecular arrangement for the functional parts of the red cell membranes calcium pump.
TL;DR: It was concluded that results obtained from the xylene adherence test were not predictive of the adherence of CNS to the hydrophobic FEP surface, and initial adherence to FEP and xylene is hampered by EP.
Abstract: The ability of 21 nonencapsulated and 15 encapsulated coagulase-negative staphylococci (CNS) to adhere to xylene in xylene-water emulsions and to fluorinated poly(ethylenepropylene) (FEP) films revealed remarkable differences. Nonencapsulated CNS strains adhered well to FEP, whereas their adherence to xylene ranged widely. Encapsulated strains with low adherence to xylene showed slight adherence to FEP. Encapsulated strains which adhered well to xylene ranged widely in their adherence to FEP. It was concluded that results obtained from the xylene adherence test were not predictive of the adherence of CNS to the hydrophobic FEP surface. The number of nonwashed, slime-producing CNS strains adhering to FEP was similar to that of washed bacteria of the same strains. Bacterial adherence to FEP was decreased when FEP films were exposed to a solution containing extracellular products (EP) obtained from a slime-producing CNS strain. Bacterial adherence to xylene also decreased when the bacterial suspensions contained EP. Apparently, initial adherence of CNS to FEP and xylene is hampered by EP. Nonencapsulated and encapsulated CNS pretreated with proteolytic enzymes failed to adhere to xylene and FEP, indicating that intact surface proteins or constituents associated with surface proteins mediated their adherence to xylene and FEP. Freeze-etch replicas of a CNS strain adhering to FEP showed a smooth, flattened area on the bacterial surface at the contact site of the bacteria with the FEP, indicating that an external layer was present at the bacterial surface.
TL;DR: The chapter proposes to develop a cocktail that is added to the bottle before blood collection to prevent the multiple enzymatic degradations that can occur during and after the withdrawal of blood.
Abstract: Publisher Summary Although the problem of artifacts originating after blood collection has been recognized, there is no general awareness of this problem among all of the workers in the field. In view of this, the chapter describes various precautionary measures for collecting blood destined for lipoprotein isolation. Studies have shown that proteolytic enzymes of different types can affect the cleavage of proapoA-I, apoA-II, apoB, and apoE; for example, the enzyme responsible for the cleavage of proapoA-I to apoA-I is present in circulation and is inhibited by ethylenediaminetetraacetic acid (EDTA). The chapter proposes to develop a cocktail that is added to the bottle before blood collection to prevent the multiple enzymatic degradations that can occur during and after the withdrawal of blood. However, proper mixing of the cocktail with the collected blood is essential, because it will ensure that all constituents have come in contact and that preventive measures have begun. The chapter concludes that the lipoprotein distribution varies from individual to individual and is a characteristic of each normolipemic subject independent of time.
TL;DR: Examples are given in this review for transmembrane processes and the role of signal peptidases of both eukaryotic and prokaryotic origin, the processing of prohormones and precursors of growth factors, protein components of blood coagulation, fibrinolysis, and of the complement system.
Abstract: The growing realization of their physiological importance has generated renewed interest in the study of proteolytic enzymes. Modern methods of protein chemistry and molecular biology have revealed new insights into the protein and gene structure of a variety of protein precursors and their processing by limited proteolysis. Examples are given in this review for transmembrane processes and the role of signal peptidases of both eukaryotic and prokaryotic origin, the processing of prohormones and precursors of growth factors, protein components of blood coagulation, fibrinolysis, and of the complement system, and a group of granulocyte proteases, including the mast cell serine proteases. The relationship of homologous domains found in many of these proteases and their zymogens to protein evolution is a recurrent theme of this discussion.
TL;DR: The presence of “beta‘’ protein in both NFT and amyloid-containing fractions suggests that it may be an important constituent of both, and cross-contamination cannot be excluded.
Abstract: Fractions enriched in neurofibrillary tangles (NFT) and amyloid fibrils were isolated from the cerebral cortex of three cases of senile dementia of the Alzheimer type Distilled water suspensions of these fractions were excluded from all pore size gels and resisted digestion with various proteolytic enzymes Formic acid/chloroform treatment of each fraction resulted in the appearance of 4,000–6,000, 15,000–17,000 and 24,000 molecular weight proteins, with concomitant diminution in the amount of excluded material at the top of each gel The 4,000–6,000 dalton band was best seen in fractions containing randomly arranged amyloid fibrils, and its amino acid composition resembled that of the recently reported “beta” protein A polyclonal antiserum to purified NFT reacted with tangles in neurons and in dystrophic neurites around plaques by immunoperoxidase staining No reaction was obtained with cerebrovascul ar or plaque core amyloid immunohistologically, or with the 4–6 kD prote in on immunoblots Cross-reactivity with the neurofibrillary lesions occurring in Pick's disease, progressive supranuclear palsy, postencephalitic Parkinsonism and dementia pugilistica was also seen Specific binding of this antiserum to the double filamentous structure was confirmed by immunoelectron microscopy Although the presence of “beta” protein in both NFT and amyloid-containing fractions suggests that it may be an important constituent of both, cross-contamination cannot be excluded
TL;DR: In vitro, elastolytic activity of bronchial secretions from patients with cystic fibrosis was significantly inhibited by eglin C and an oxidation-resistant variant of alpha 1-PI, both compounds currently produced by recombinant DNA technology.
Abstract: Large amounts of free granulocyte elastase (GE), an enzyme capable of mediating airway damage, have been found in bronchial secretions of patients with cystic fibrosis who are infected with Pseudomonas aeruginosa. This finding indicates an imbalance between GE and its antiproteases, alpha 1-proteinase inhibitor (alpha 1-PI) and bronchial mucosal inhibitor (BMI), in the airways of these individuals. The effect of intravenous antimicrobial treatment against P. aeruginosa on activity and concentration of GE, BMI, and alpha 1-PI was evaluated in 30 treatment courses of 20 patients with cystic fibrosis. Although sputum volume and level of immunoreactive GE decreased and concentrations of alpha 1-PI and BMI increased significantly (P less than .05), a high level of free GE persisted. No active alpha 1-PI and BMI were detectable after treatment. High levels of GE correlated with a poor pulmonary condition (rs = .98, P less than .001). In vitro, elastolytic activity of bronchial secretions from patients with cystic fibrosis was significantly inhibited by eglin C and an oxidation-resistant variant of alpha 1-PI, both compounds currently produced by recombinant DNA technology.
TL;DR: The hemagglutinating factor of Bacteroides gingivalis was prepared from the supernatant of a 5-day diffusate broth culture by ammonium sulfate precipitation and column chromatography with a hydrophobic column of Phenyl-Sepharose CL-4B, DEAE-Sephadex A-50, and SephadeX G-100 gel filtration.
Abstract: The hemagglutinating factor (hemagglutinin) of Bacteroides gingivalis was prepared from the supernatant of a 5-day diffusate broth culture by ammonium sulfate precipitation and column chromatography with a hydrophobic column of Phenyl-Sepharose CL-4B, DEAE-Sephadex A-50, and Sephadex G-100 gel filtration. The hemagglutinating activity of the preparation was 53.3 times higher than that of ammonium sulfate precipitate. In electron microphotographs, hemagglutinin appears to have a vesicle or tubelike structure. The hemagglutinating activity of intact cells was completely destroyed by heating at 100 degrees C for 10 min, but the activity of extracted hemagglutinin was heat stable. The activity of hemagglutinin was inhibited by L-arginine and L-lysine and partially inhibited by phospholipase D, but it was not affected by proteolytic enzymes, neuraminidase, hyaluronidase, lipase, phospholipase A and C, or sugars. The B. gingivalis hemagglutinin appeared to be comprised mainly of a 40,000-molecular-weight material. The Fab fragment of immunoglobulin G prepared from rabbit antiserum to whole cells of B. gingivalis and monoclonal antibody against the hemagglutinin bound to the cell surface and inhibited the hemagglutinating activity of both the cells and the purified hemagglutinin.
TL;DR: It is proposed that a critical step is the interaction of bacterial antigens with inflammatory cells, resulting in the production of a cytokine, interleukin-1, whose production may not be dependent on a specific microbial flora but may be triggered by a number of organisms.
Abstract: Much of the connective tissue degradation that takes place in periodontal diseases is mediated by proteolytic enzymes. Previous studies have focused on the action of proteinases released by invading polymorphonuclear neutrophils and macrophages, and bacterial enzymes. In view of recent work establishing that resident connective tissue cells can be induced by cytokines to bring about the destruction of their own matrix, we propose a new hypothesis. In this we envisage that a critical step is the interaction of bacterial antigens with inflammatory cells, resulting in the production of a cytokine, interleukin-1. Our interpretation of in vitro evidence is that the loss of connective tissue attachment and bone matrix resorption in periodontal diseases is mediated by metalloproteinases such as collagenase and stromelysin released by cells of the periodontium. Such proteolytic destruction can be induced by interleukin-1, whose production may not be dependent on a specific microbial flora but may be triggered by a number of organisms. It is now clear that interleukin-1 has multiple actions on both immune and non-immune cells; these include the induction of lymphocyte differentiation and proliferation and the stimulation of bone and cartilage resorption, and prostaglandin and metalloproteinase synthesis by connective tissues. It seems likely that further knowledge about the production and function of this cytokine will have an increasing impact in many diseases that involve resorption, particularly since interleukin-1-like molecules can be produced by cell types other than monocytes/macrophages, including keratinocytes and fibroblasts.
TL;DR: Results indicate that the presence of the 6 kDa apoprotein can account for some of the essential physical and biological characteristics of pulmonary surfactant.
Abstract: Lipid extracts of bovine pulmonary surfactant containing the 6 kDa apoprotein, but lacking the 35 kDa apoprotein, can mimic the essential characteristics of pulmonary surfactant on a pulsating-bubble surfactometer. Reconstituted surfactant can be produced by combining silicic acid fractions containing 6 kDa apoprotein and phosphatidylglycerol with phosphatidylcholine. Treatment of the protein-containing fraction with proteolytic enzymes abolishes its efficacy. These results indicate that the presence of the 6 kDa apoprotein can account for some of the essential physical and biological characteristics of pulmonary surfactant. Immunodiffusion studies indicate that, contrary to earlier suggestions, the 6 kDa apoprotein is not structurally related to the major surfactant apoprotein that has a molecular mass of 35 kDa.
TL;DR: The idea that serum deprivation and/or removal of the cumulus cells during oocyte maturation in vitro results in an alteration of the zona pellucida that is manifested as an increased resistance to proteolytic digestion and sperm penetration is supported.
Abstract: The effects of serum and cumulus cells during oocyte maturation in vitro on subsequent oocyte fertilizability and zona pellucida digestability have been examined. Cumulus cell-enclosed oocytes were cultured 15–16 hours in medium containing bovine serum albumin plus varying concentrations of fetal bovine serum (FBS). When the serum concentration was decreased incrementally from 5% to 0%, resistance to chymotrypsin digestion increased accordingly in a dose-dependent manner. The zona pellucida digestion time for oocytes matured in serum-free medium was increased nearly 500% above that for control oocytes matured in 5% FBS. Also, fertilization was decreased as serum concentration was lowered; the fertilization percentage for oocytes matured in serum-free serum was reduced by over 90%. This loss of fertilizability was correlated with an absence of sperm within the vitellus following insemination of matured ova. Increasing the serum concentration from 5% to 10% had no effect on fertilization or zona pellucida digestion time. Serum deprivation, even for a short period of time, at the onset of culture significantly reduced the fertilizability of cumulus cell-enclosed oocytes and increased the zona pellucida digestion time. Removal of serum after oocyte maturation in vitro had little effect. The presence of an intact cumulus oophorus during maturation in vitro was important in the maintenance of fertilizability and zona digestability. These data support the idea that serum deprivation and/or removal of the cumulus cells during oocyte maturation in vitro results in an alteration of the zona pellucida that is manifested as an increased resistance to proteolytic digestion and sperm penetration.
TL;DR: Methods are described for the mechanical isolation of endothelial cells from bovine pulmonary artery by gently scraping the luminal surface with a scalpel, and treatment with proteolytic enzymes is thus avoided at isolation and during culture and subculture.
Abstract: Methods are described for the mechanical isolation of endothelial cells from bovine pulmonary artery (or any large vessel) by gently scraping the luminal surface with a scalpel. Monolayers of endothelial cells are cultured from these scrapings. Any contaminating smooth muscle cells or fibroblasts are ringed with a grease pencil and not removed when the cells are subcultured. Both purification and subculture are effected by scraping with a rubber policeman. Treatment with proteolytic enzymes is thus avoided at isolation and during culture and subculture.
TL;DR: HT29-D4 and HT9-D9 cells can be maintained permanently in a differentiated state in a glucose-free medium and were able to form domes at confluency, demonstrating for the first time that proteolytic enzymes are potent inducers of dome formation.
Abstract: The replacement of glucose by galactose in the culture medium resulted in partial structural and functional enterocytic differentiation of HT29 cells. In order to characterize populations of homogeneously differentiated HT29 cells we have selected two clonal cell lines HT29-D4 and HT29-D9 with the following functional and structural characteristics when grown in a galactose-containing medium: the two clonal cell populations were permanently morphologically differentiated as shown by the presence of mature junctional complexes and a well-organized brush border (especially for HT29-D4 cells); HT29-D4 and HT29-D9 cells were able to form domes early in confluency, which indicated a functional state of differentiation; the process of differentiation was fully reversible when glucose was added to the culture medium. The induction of domes was investigated in these two cell populations and we demonstrated for the first time that proteolytic enzymes are potent inducers of dome formation. The architecture of domes either obtained spontaneously or induced by proteolytic enzymes was not maintained in the presence of ouabain (a specific inhibitor of the Na+/K+-ATPase). In conclusion, HT29-D4 and HT29-D9 cells can be maintained permanently in a differentiated state in a glucose-free medium and were able to form domes at confluency. The observation that proteolytic enzymes were able to induce dome formation can help in the comprehension of the mechanism involved in the establishment of the differentiated state.
TL;DR: Casein kinase II activity in developing mouse embryos shows a 3-4-fold activity increase at day 12 of gestation and increased phosphorylation of a 110-kDa protein is observed, indicating that the protein might be a CKII-specific substrate.
Abstract: Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3–4-fold activity increase at day 12 of gestation. Together with the CKII activity, increased phosphorylation of a 110-kDa protein is observed. Treatment of the embryo extracts with heparin, a highly specific inhibitor of CKII activity, results in a drastic reduction of the 110-kDa protein phosphorylation indicating that the protein might be a CKII-specific substrate. Rapidly proliferating mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP-and cGMP-dependent protein kinases) only show a basal level of enzyme activity with minor alterations throughout the different stages of embryogenesis investigated.
TL;DR: It is speculated that these sulfated glycans are essential for maintaining the structural integrity of the tunic, in analogy with the glycosaminoglycans of vertebrate connective tissues.
TL;DR: It was concluded that low Mr proteins are specifically stored in ovine ceroid lipofuscinosis and that the ceroids may result from inherited defects in lysosomal protein catabolism.
TL;DR: It has been suggested, although not confirmed, that deficiency in serum protease inhibiting capacity could be correlated with susceptibility to periodontal disease, and bacteria-derived, antiproteolytic short peptides may prove to be useful in pharmacological control of tissue destruction at inflammatory sites.
Abstract: This article reviews the current knowledge of the sources, function and interactions of proteolytic enzymes and their inhibitors in chronic inflammatory periodontal disease. Proteolytic tissue degradation is a typical phenomenon in chronic inflammatory periodontal disease. The proteolytic enzymes can be both host- and bacteria-derived. The proteases of the inflammatory cells are aimed for digestion of bacteria, enhanced locomotion through connective tissue, demarcation of the site of infection and tissue remodeling. Uncontrolled release of proteases in inflammation causes self-digestion and tissue destruction. The potential of the bacterial proteases in degradation of connective tissue is not yet known. Biochemical and immunologic mediators of inflammation are released by proteolytic reactions. Immunoglobulin-cleaving proteases present a specific mechanism in perturbation of host defenses. The 2 main protease inhibitors in serum, alpha-1-antitrypsin and alpha-2-macroglobulin, are also present in the gingival tissue fluid guarding the function of proteases. It has been suggested, although not confirmed, that deficiency in serum protease inhibiting capacity could be correlated with susceptibility to periodontal disease. Mucous secretions contain local low molecular weight protease inhibitors, but their possible role in saliva is not known. Bacteria-derived, antiproteolytic short peptides may prove to be useful in pharmacological control of tissue destruction at inflammatory sites.