Latency of Infection in Unripe Avocados by Colletotrichum gloeosporioides is Not Due to Inadequate Enzyme Potential

Colletotrichum gloeosporioides produced exo-pectin lyase and protease in a) liquid cultures with incorporated washed cell wall material from unripe or ripe avocado and b) autoclaved immature fruit. The activity of exo-pectin lyase and protease produced in liquid cultures incorporating washed cell walls from immature fruits was almost the same as when washed cell walls from ripe fruits were incorporated. Ripe fruit tissue rotted by the fungus contained exo-pectin lyase, endo-polygalacturonase (endo-PG) and protease. The endo-PG was found to be endogenous to avocado fruit, and had a pH optimum of 5.5. The pH optima of exo-pectin lyase and protease were 8.5 and 7.5 respectively in all three enzyme preparations. All these enzyme preparations completely macerated avocado fruit tissue discs in vitro in less than 3 h of incubation but not potato tuber discs. Neither immature nor ripe fruit contained substances, proteinaceous or otherwise, which could inhibit the exo-pectin lyase or protease activity of these preparations. The results indicated that C. gloeosporioides possesses sufficient enzyme potential to invade cell walls of unripe fruit and that the fruit tissue does not have a mechanism to inactivate such enzymes.     

Expression of protease and protease-inhibitory activity in human mononuclear leukocyte cultures : Effect of conA stimulation

Trypsin-activated protease activity was observed in the supernatants of conA-treated unfractionated or monocyte-depleted cultures at 1 h of incubation and trypsin-inhibitory activity was detected at 24 h. In contrast, supernatants from wheat germ agglutinin (WGA)-treated cultures or untreated cultures exhibited trypsin-inhibitory activity at 1 h and trypsinactivated protease activity after 24 h. The expression of proteases and protease inhibitors may be early events that occur during the activation of quiescent lymphocytes to a proliferative state.     

Rat hepatocyte primary cultures. IV. Maintenance in defined medium and the role of production of plasminogen activator and other proteases

Primary cultures of rat hepatocytes survived well for up to 4 days in defined medium in the presence of dexamethasone but not in its absence. The loss of viability was accompanied by a loss of ultrastructural features characteristic of hepatocytes. The cultures began producing plasminogen activator and a neutral protease after 24 hr in culture. Dexamethasone inhibited the production of both of these substances. The deterioration of the cultures appeared not to be related to plasminogen activator, but prolongation of survival by a variety of protease inhibitors suggested that the neutral protease might contribute to deterioration.     

The effects of serine protease inhibitors on morphological differentiation of murine neuroblastoma cells (NB 15)

Morphological differentiation of neuroblastoma cells (NB15) was induced by cAMP effectors in the presence and absence of serine protease inhibitors. In all conditions tested, the percent differentiation was inhibited by protease inhibitors antipain, diisopropylfluorophosphate (DFP), leupeptin, and soybean trypsin inhibitor (SBTI). The level of morphological differentiation obtained in medium containing fetal calf serum was significantly less than the percent differentiation obtained with serum-free medium alone, so serum-free medium was the principal method of induction and comparisons were made to control uninduced cultures or cultures induced with the phospho-diesterase inhibitor R020–1724. Secreted or cell surface caseinolytic protease activity was higher in differentiating cells than in control cultures and was inhibited by the serine protease inhibitors. The effects of the protease inhibitors on growth and differentiation are discussed.     

Rat hepatocyte primary cultures

Summary Primary cultures of rat hepatocytes survived well for up to 4 days in defined medium in the presence of dexamethasone but not in its absence. The loss of viability was accompanied by a loss of ultrastructural features characteristic of hepatocytes. The cultures began producing plasminogen activator and a neutral protease after 24 hr in culture. Dexamethasone inhibited the production of both of these substances. The deterioration of the cultures appeared not to be related to plasminogen activator, but prolongation of survival by a variety of protease inhibitors suggested that the neutral protease might contribute to deterioration. Dr. Goldblatt was supported by Grant No. SG-87 from the American Cancer Society as an American Cancer Society Scholar while on sabbatical leave from the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut. This study was supported by Contracts NO1-CP-55705 from the National Cancer Institute and 68-02-2483 from the Environmental Protection Agency.     

Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes

Bovine pancreatic trypsin-inhibitor (bPTI) is required for survival of adult rat hepatocytes for more than 2 days in primary cultures in serum-free medium. Of the various protease inhibitors tested, all trypsin inhibitors increased the survival of rat hepatocytes in serum-free medium, their potencies being in the order bPTI greater than alpha 2-plasmin inhibitor greater than leupeptin greater than soybean trypsin inhibitor greater than alpha 1-antitrypsin = alpha 2-macroglobulin. Elastatinal, a specific inhibitor of elastase, was also effective. bPTI did not inhibit the degradation of proteins with short or long lives, suggesting that it did not increase the survival of hepatocytes by inhibiting cellular protein degradation. alpha 2-Plasmin inhibitor immobilized on Sepharose 4B caused dose-dependent increase in survival. Plasma membranes purified from adult rat liver had significant protease activity, about 80% of which was sensitive to bPTI, alpha 2-plasmin inhibitor and leupeptin. From its specificity for substrates and sensitivity to inhibitors, the membrane-bound protease was characterized as a trypsin-like protease. The effects of various inhibitors on the membrane-bound protease correlated well with their abilities to increase survival of rat hepatocytes. Therefore, it seems that bPTI acts on the cell surface and increases hepatocyte survival in serum-free cultures by inhibiting a trypsin-like protease associated with the plasma membranes.     

Rapid release of protease inhibitors from soybeans: immunochemical quantitation and parallels with lectins

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.     

The effects of bestatin, a microbial aminopeptidase inhibitor, on epidermal growth factor-induced DNA synthesis and cell division in primary cultured hepatocytes of rats

We investigated the effects of microbial protease inhibitors, in particular the aminopeptidase inhibitor bestatin, on DNA synthesis and cell division induced by epidermal growth factor (EGF) in hepatocytes. Although bestatin did not significantly affect binding of EGF to hepatocytes, it inhibited EGF-induced DNA synthesis and cell division. DNA synthesis in rat hepatocytes was maximal 24-26 h after EGF addition to the medium. The time required for maximal DNA synthesis was not affected if bestatin was removed less than 12 h after addition, but synthesis was partially inhibited if bestatin was added to the medium several hours after EGF addition, depending on the time of bestatin addition. Our results suggest that bestatin arrests the new cell cycle induced by EGF at about 12 h after the initiation. Considering also our results obtained by employing other protease inhibitors, we concluded that specific proteases play important roles in hepatocyte DNA synthesis and cell division induced by EGF.     

Phorbol ester-induced morphological changes in transformed chick fibroblasts: evidence for direct catalytic involvement of plasminogen activator.

The tumor promoter phorbol myristate acetate (PMA) induces the production of the serine protease plasminogen activator (PA) in cultures of normal chick embryo fibroblasts (CEF) and synergistically enhances PA production in Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF). Following PMA treatment of serum-free RSVCEF cultures, PA induction is accompanied by distinct morphological changes, including enhanced cell clustering and the formation of dense cellular aggregates. These alterations in the morphology of the PMA-treated transformed cells are inhibited by several protease inhibitors, including leupeptin, NPGB, SBTI, benzamidine and DFP, the specific inhibitor of serine enzymes. A number of protease inhibitors are ineffective in preventing the PMA-induced morphological changes; these include inhibitors of trypsin, chymotrypsin, elastase, thrombin and, most importantly, plasmin. The use of a fluorescent substrate to assay PA directly demonstrated that the pattern of inhibiton of PA activity correlates exactly with the inhibition of morphological changes. The of 3H-DFP to label and characterize serine zymes in the culture fluid from PMA-treated cells further indicated that PA is the serine protease responsible for the morphological changes. Thus PA itself can catalytically alter cellular behavior in culture independent of plasminogen, until not its only known natural substrate.     

Luteinizing hormone and cyclic AMP-mediated induction of microsomal cytochrome P-450 enzymes in cultured mouse Leydig cells

Treatment of mouse Leydig cell cultures with luteinizing hormone (LH) or with 8-bromo-cAMP (8-Br-cAMP) for 5 days elicited a dose- and time-dependent increase in the microsomal cytochrome P-450 enzyme activities. 17 alpha-Hydroxylase and C17-20 lyase as well as a parallel increase in testosterone production. Reduction of the oxygen tension from 19 to 1% resulted in a greater increase in enzyme activity. Induction of microsomal cytochrome P-450 activities was 35 to 50% greater with 8-Br-cAMP than with LH and the increase in C17-20 lyase activity was 4-fold greater than that of 17 alpha-hydroxylase. Maximal induction of P-450 enzyme activities was observed between 3 and 5 days of continual treatment with 8-Br-cAMP or LH. Removal of 8-Br-cAMP from the culture medium inhibited any further increase in C17-20 lyase activity and testosterone production. The role of protein synthesis in the induction process was investigated by incubating Leydig cell cultures with and without cycloheximide between 24 and 48 h of treatment with 8-Br-cAMP. Cycloheximide completely inhibited the induction of C17-20 lyase activity and the increase in testosterone production. After removal of the inhibitor, cultures responded in a manner that paralleled induction in cultures that had not been treated with cycloheximide. In both cases, a 24-h lag period occurred prior to an increase in cytochrome P-450 activity. These data suggest that the increase in microsomal cytochrome P-450 activities represents an increase in enzyme synthesis and, furthermore, that reduction of oxygen tension decreases degradation of newly synthesized Leydig cell microsomal cytochrome P-450 activities as recently reported (Quinn, P.G., and Payne, A.H. (1984) J. Biol. Chem. 259, 4130-4135).     

Viral therapy: prospects for protease inhibitors

Antiviral activities of known protease inhibitors were assayed in virus-infected cell cultures. Some members of the cystatin superfamily, in particular chicken cystatin, were able to block virus replication. In a binding assay, using purified components, chicken and human cystatin were able to bind poliovirus protease with affinities which were reflected in their relative antiviral potencies. Prospects for application of protease inhibitors in clinical viral infections are discussed.     

Protease production by Streptomyces thermovulgaris grown on rapemeal-derived media

A range of actinomycete species was tested for their ability to grow on particulate and particle-free rapeseed meal-derived media. Streptomycetes grew on both types of medium and produced a number of extracellular enzymes. Highest activities of protease were produced by Streptomyces thermovulgaris and reflected the high available protein content of rapemeal. Enzyme production and growth were analysed in fermentor-grown batch cultures of S. thermovulgaris using the particle-free rapemeal broth termed medium B. Growth was biphasic and the majority of the protease was produced during the second slower phase. Analysis of the protease as azocaseinase activity revealed a high degree of thermostability in the presence of calcium such that approximately 20% of the activity remained after incubation at 70°C for 24 h. Gel filtration suggested that S. thermovulgaris synthesized more than one kind of protease and this was confirmed by using specific peptide substrates and inhibitors which revealed the presence of distinct serine and metallo-type enzymes.     

Serum protease inhibitors promote anchorage-independent growth of transformed cells in serum-free medium.

The effect of three serum serine protease inhibitors on the serum-free agar growth of an SV40-transformed 3T3 cell line was investigated. Antithrombin III, alpha-2-macroglobulin and alpha-1-antitrypsin were found to potently stimulate colony growth in a semisolid medium because of their anti-proteolytic properties. These results indicate that protease inhibitors can facilitate tumor cell growth in serum-free agar cultures and suggest that the stimulatory effect of serum on the growth of certain transformed cells in agar may at least partially be due to the high levels of protease inhibitors found in serum.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.     

Phenol metabolism, phytoalexins, and respiration in potato tuber tissue treated with Fatty Acid

Potato (solanum tuberosum L. cv Katahdin) tuber discs treated with arachidonic acid become necrotic and accumulate sesquiterpenoid phytoalexins. The arachidonic acid also causes increases in both phenylalanine ammonia lyase and lignin, but no change in total alcohol-soluble phenols. Linoleic acid does not alter any of these parameters. A high concentration of nonanoic acid promotes both necrosis and accumulation of low levels of phytoalexins, but decreased levels of phenols, phenylalanine ammonia lyase, and lignin. The respiration of the control discs and those treated with linoleic acid declines by 24 hours after treatment, but the respiration of arachidonic acid-treated discs remains constant for at least 48 hours.     

Pectinase Production by Bacteria Associated with Improved Preservative Permeability in Sitka Spruce: Synthesis and Secretion of Polygalacturonate Lyase by Cytophaga johnsonii

Cytophaga johnsonii synthesized a polygalacturonate lyase which produced random cleavage of galacturonic acid polymers. No pectin methyl-esterase or hydrolytic pectinase activities could be detected in cultures of the organism. Polygalacturonate lyase synthesis was inducible and also subject to repression by glucose and other compounds. Galacturonic acid was the most effective inducer; lower activities were obtained with citrus pectin, polygalacturonic and polypectic acids. Glucose repression of lyase synthesis was not alleviated by 5 mM-adenosine-3'.5'-cyclic-monophosphate. Enzyme production was growth-linked and ceased when batch cultures entered the stationary phase. In steady-state chemostat cultures lyase activity was maximal at a dilution rate ( D ) of 0.19 h^-1. Polygalacturonate lyase was both cell-bound and free in the supernatant medium. The proportion of free enzyme increased throughout the batch growth cycle and in chemostat cultures over 70% of the activity was cell free at dilution rates below 0.05 h^-1.     

Properties of adenosine monophosphate deaminase of Candida albicans.

Adenosine monophosphate deaminase (AMPD; EC 3.5.4.6) catalyses the hydrolysis of adenosine monophosphate (AMP) to commensurate amounts of inosine monophosphate (IMP) and ammonia. The production of AMP deaminase in Candida albicans was measured in Lee's medium grown cultures. The highest AMPD activity was observed at 24 h of growth. The enzyme had an optimum pH and temperature at 6-7 and 28 degrees C, respectively. This enzyme was inhibited under iron-limited growth conditions as well as by protease inhibitors. The AMPD of C. albicans showed a moderate increase in activity when cultures were grown in the presence of the divalent cations Mg2+, Ca2+, and Zn2+. Moreover, ADP, ATP, adenine, adenosine, deoxyribose and hypoxanthine increased the enzyme activity. Cultures grown in trypticase soy broth exhibited maximum AMPD activity compared with those grown in Sabouraud dextrose broth or Lee's medium.