Effects of proteinase inhibitors on the cutaneous lesion ofSporothrix schenckii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin. On the other hand, proteinase II is an aspartic proteinase, inhibited by pepstatin. The addition of either pepstatin or chymostatin to the culture medium did not inhibit cell growth, however the addition of both inhibitors strongly inhibited fungal growth. Accordingly, this suggested that extracellular proteinases play an important role in cell growth and that such cell growth may be suppressed if these proteinases are inhibited. In order to substantiate this speculation in sporotrichosis, the effects of proteinase inhibitors on the cutaneous lesions of mice were studied. Ointments containing 0.1% chymostatin, 0.1% pepstatin and 0.1% chymostatin-0.1% pepstatin were applied twice daily on the inoculation sites of hairless mouse skin, and the time courses of the lesions examined. The inhibitory effect in vivo onS. schenckii was similar to that demonstrated in our previous in vitro study. Compared to the control, the time course curve of the number of nodules present after the application of either pepstatin or chymostatin was slightly suppressed. The application of both pepstatin and chymostatin, however, strongly suppressed nodule formation. This study not only confirmed the role of 2 proteinases ofS, schenckii for fungal growth in vivo, but also may lead to their use as new topical therapeutic agents.     

Hormonal control of hyaluronic acid production in fibroblasts and its relation to nucleic acid and protein synthesis.

Whole serum and elevated pH previously had been found to stimulate both cell multiplication and hyaluronic acid production by chick embryo fibroblasts in culture. In a study to determine whether cell multiplication and hyaluronic acid production both respond to a single well-defined substance, insulin was found to stimulate, and cortisol to inhibit both processes coordinately. It appears, therefore, that multiplication and differentiated function in fibroblasts respond to a common underlying regulatory signal. Inhibition of ribosomal RNA synthesis by actinomycin D does not prevent serum stimulation of hyaluronic acid production, but inhibition of total RNA synthesis does. If total RNA synthesis is inhibited only after the hyaluronic acid production has reached a new high level, it continues at that level for the next five hours. The stimulatory treatment causes an increase in the activity of the enzyme hyaluronate synthetase. Inhibition of protein synthesis prevents any increase in hyaluronic acid production, and reduces the basal level of production. Reduction of the availability of Mg2+ in the medium coordinately inhibits DNA synthesis and hyaluronic acid production. The results are discussed in the light of a model for coordinate control growth and metabolism based on the availability of Mg2+.     

A serine proteinase inhibitor produced by an HTLV I virus-transformed human T lymphocyte line

A previous report from our laboratory indicated that a proteinase inhibitor is produced by rabbit T lymphocytes. We now report that a human T cell line, C91/PL, produces a proteinase inhibitor which inhibits the enzymatic activity of trypsin and kallikrein. This newly identified proteinase inhibitor (LPI 1) did not inhibit the enzymatic activity of four other serine proteinases (thrombin, plasmin, chymotrypsin, or pancreatic elastase), a thiol proteinase (papain), or a carboxyl proteinase (pepsin). Active synthesis of LPI 1 by the C91/PL cell line was shown by the appearance of similar levels of inhibitory activity in sequential cell supernatants, lack of appearance of inhibitor in supernatants of cells killed by heat or sodium azide or of viable cells in the presence of cyclohexamide, and incorporation of a radiolabeled amino acid into newly synthesized inhibitor. Although both the inhibitor of rabbit origin and of human origin are proteins produced by T cells and have similar inhibitory specificity, important differences were observed: LPI 1 is sensitive to boiling and the two inhibitors migrate differently upon electrophoresis in substrate-containing polyacrylamide gel. Furthermore, LPI 1 was produced by a cell line of the T4 phenotype which had been established by in vitro viral transformation of human cord blood lymphocytes with HTLV 1 whereas the inhibitor of rabbit origin was produced by normal splenic T cells. Three other human T cell lines of the T4 phenotype, MOLT-13, KE-37, and HPB-ALL, from patients with acute lymphoblastic leukemia did not produce a proteinase inhibitor. Thus, the production of proteinase inhibitors does not appear to be a general characteristic of human T cell lines nor of the T4 subset. Proteinase inhibitors produced by T cells may have an immunoregulatory role in proteinase-mediated physiological processes.     

江浙蝮蛇毒精氨酸酯酶Agkihpin的研究Ⅱ.对鼻咽癌细胞活力、增殖、迁移和细胞形态的影响

目的：将江浙蝮蛇粗毒中新分离纯化的一种精氨酸酯酶Agkihpin应用于鼻咽癌细胞LXC的体外培养,观察Agkihpin对鼻咽癌细胞活力、增殖、迁移和细胞形态的影响,以期探索治疗鼻咽癌的新方法、新药物。方法：将不同剂量的Agkihpin加入细胞培养液中,用四甲基偶氮唑（MTT）法分析细胞活力,细胞计数法观察和分析细胞增殖和细胞迁移。结果：一定剂量的Agkihpin可抑制LXC的细胞活力、增殖、迁移,并可改变细胞形态和杀伤LXC,且剂量越大抑制、杀伤和细胞形态改变越明显。结论：Agkihpin对鼻咽癌的治疗具有潜在的重要意义,Agkihpin具有作为抗鼻咽癌新药物的开发潜力。     

Changes in sensitivity of human tumour cells to growth inhibition by proteinase inhibitors.

Inhibition of a cell-surface proteinase can inhibit the growth of many normal human cell types in culture. Some tumour cells are also sensitive to proteinase inhibitors, but others are resistant, and continue to grow in the presence of these inhibitors. Here we describe two human tumour cell lines which convert from the sensitive to the resistant state. In one case, the conversion occurs during routine passaging, but, in the other, it is determined by growth conditions, and is reversible.     

Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor).

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

Suppression of snake-venom cardiotoxin-induced cardiomyocyte degeneration by blockage of Ca2+ influx or inhibition of non-lysosomal proteinases.

The incubation of 10(5) single neonatal rat cardiomyocytes with 1 microM-cardiotoxin in a bath medium, Tyrode solution in the presence of 1 mM-Ca2+, at 37 degrees C evoked the following chain of events. Firstly, there appeared a latent period of about 10 min during which the cells behaved normally. Neither lactate dehydrogenase nor ATP leaked from the cells. Cytosolic free Ca2+ increased considerably, as measured by the fluorescence intensity of fura-2-Ca2+ complex. At the same time a large portion of endogenous ATP was depleted. Secondly, after the latent period, the cell beating became irregular and eventually stopped. Thirdly, blebs appeared on the cell surface, leading to cell degeneration. If, before the appearance of blebs, the cells were washed with the bath medium exhaustively or incubated in the presence of the toxin antibody, cytosolic free Ca2+ and endogenous ATP returned to normal levels and cells resumed regular beating. Preincubation of the cells with 3.75 microM-flunarizine or 3.75 microM-diltiazem (both are Ca2+ antagonists), or 1.5 microM-fura-2 acetoxymethyl ester (a chelate for Ca2+), or 200 microM-leupeptin or 50 microM-antipain (both are proteinase inhibitors) considerably suppressed the toxin's ability to degenerate the cells. On the other hand, lysosomal proteinase inhibitor, autophage inhibitor, serine proteinase inhibitor, phospholipase inhibitor and calmodulin antagonist did not inhibit the toxin's activity. The results suggest that the toxin may act on the extracellular surface of intact cardiomyocytes to increase cytosolic free Ca2+. The subsequent cell degeneration may result from the activation of a Ca2+-dependent non-lysosomal proteolytic system.     

Differential inhibition of rat mast cell proteinase I and II by members of the alpha-1-proteinase inhibitor family of serine proteinase inhibitors

Rat mast cell proteinase II (RMCP II) from mucosal mast cells was titrated into rat serum, and the resulting serine proteinase inhibitor (serpin)-enzyme complex was purified by affinity chromatography on anti-RMCP II-Sepharose 4B and by Mono-Q anion-exchange. The purified complex was used to raise polyclonal antibodies which, after cross-absorption against RMCP II-Sepharose 4B, were specific for serpin and were used to affinity purify two rat serpin molecules (RSI and RSII) that inhibit RMCP II in rat serum. The kinetic constants characterizing the interaction between RMCP II and RSI and RSII are ka, 2.2 x 10(5) and 1.65 x 10(5) M-1 s-1, respectively; Ki, 3.6 x 10(-10) and 1.0 x 10(-9) M; and kd, 7.9 x 10(-5) and 1.65 x 10(-4) s-1. Amino-terminal sequence analysis indicated that RSI and RSII are distinct, differing at the amino-terminal residues, and are products of the rat Spi-1 locus. Rat mast cell proteinase I (RMCP I) from connective tissue mast cells cleaved both RSI and RSII and was not inhibited.     

The effect of endogeneous proteinase inhibitors on the prophenoloxidase activating enzyme,a serine proteinase from crayfish haemocytes

Three proteinase inhibitors have so far been isolated and purified from crayfish haemolymph. One of these, isolated from crayfish plasma, namely a trypsin inhibitor with a molecular mass of 155 kDa was found to inhibit a serine proteinase, ppA, which is involved in the activation of prophenoloxidase, and is localized in the haemocytes. Another high molecular mass proteinase inhibitor, an α₂-macroglobulin from crayfish plasma, which is a dimer of 190 kDa-subunits, was only inhibitory towards ppA to a lesser extent. A 23 kDa subtilisin inhibitor, purified from haemocytes, did not have any effect on the serine proteinase.We suggest that mainly the trypsin inhibitor, but to some extent also the α₂-macroglobulin, are important in the regulation of the prophenoloxidase activating cascade, as they both inhibit ppA, which in its active form has been shown to mediate prophenoloxidase activation.     

Identification of a soluble enzyme from C3H/10T1/2 cells which is inhibited by the Bowman-Birk proteinase inhibitor

The anticarcinogenic Bowman-Birk proteinase inhibitor (BBI) inhibits a 70-kDa serine proteinase in C3H/10T1/2 transformed fibroblasts. Two serine proteinases, the proline endopeptidase and a novel neutral proteolytic activity, both having a mass of approximately 70-kDa, were isolated from the cytoplasm of C3H/10T1/2 cells. BBI did not inhibit diisopropylfluorophosphate binding to the proline endopeptidase or its ability to hydrolyze peptides. However, BBI blocked the binding of diisopropylfluorophosphate and inhibited the cleavage of peptides by the novel cytoplasmic enzyme. Thus BBI does not inhibit the proline endopeptidase but another soluble 70-kDa serine proteinase from C3H/10T1/2 cells.     

High molecular weight chitosan and sodium alginate effect on secretory acid proteinase of Candida albicans

The effect of high molecular weight chitosan (HMWCh) and sodium alginate (NaAL) on acid proteinase secretion of Candida albicans (one of culture collection and five isolates) was evaluated. The secretion of acid proteinase was induced in the presence and the absence of these polymers in different concentrations and their enzymatic activity was determined. HMWCh and NaAL significantly diminished the enzymatic activity (>76% for the collection strains and > 89% for the isolates, p < 0.05). HMWCh did not modify protein concentrations, but NaAL did. It can be concluded that both polymers can inhibit the proteinase activity of Candida albicans.     

Involvement of arabinogalactan proteins in the control of cell proliferation of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> suspension cultures

Arabinogalactan proteins (AGPs) secreted by zucchini squash (Cucurbita pepo L.) cell cultures into the medium are implicated in cell proliferation. Conditioned medium derived from cell suspensions of squash cultivar Dundoo could enhance multiplication rate of slow-growing cell line Cx3005. To examine the role of AGPs, a precipitation assay was performed using Yariv reagent which binds selectively to AGPs. This AGP precipitation as well as proteinase application arrested cell division. However, chitinase treatment successfully increased embryogenic callus mass. A growth promotion was also obtained by arabinogalactan addition to the culture medium. Immunoblotting analysis using the MAC 207 anti-AGP monoclonal antibody showed high AGP expression in Dundoo cell cultures.     

Plasmodium falciparum: Differential Sensitivity In Vitro to E-64 (Cysteine Protease Inhibitor) and Pepstatin A (Aspartyl Protease Inhibitor)

ABSTRACT We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 μM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 μM) or 12 h at the inhibitory concentration 50% (12 μM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 μM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

How does interferon inhibit tumour growth?

Interferon can inhibit tumour growth in experimental animals and in some patients with benign and malignant tumours. There is experimental evidence to suggest that several mechanisms may be involved: a direct effect on the tumor or an indirect effect via the host, or both. Thus, interferon may slow the rate of tumour cell multiplication and this may lead to cell death. Interferon may induce changes in the cell surface rendering tumour cells more sensitive to host defence mechanisms. Interferon may induce reversion in the phenotype of tumour cells. Interferon may stimulate specific and non-specific humoral and cellular host mechanisms. The relative importance of these different effects of interferon may vary depending on the host and the particular tumour.     

Heterotrimeric G protein mediated regulation of proteinase production in Aspergillus nidulans

Extracellular proteinase production induced by carbon starvation was studied in a series of heterotrimeric G protein signaling pathway mutants of Aspergillus nidulans. All the mutants tested--including deltafadA (Galpha), deltasfaD (Gbeta), deltagpgA (Ggamma) and deltasfgA (regulator of FadA signaling)--showed an elevated proteinase production after glucose depletion. Our results strongly support the view that during growth, FadA/SfaD/GpgA G protein signaling inhibits proteinase production via both Galpha and Gbetagamma subunits, and all conditions, which are not sufficient to support vegetative growth and, hence, inhibit this type of G protein signaling, elevate extracellular proteinase activities.     

Insulin-degrading neutral cysteine proteinase activity of adipose tissue and liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats

The activity of the insulin-degrading enzyme neutral cysteine proteinase (EC 3.4.22.11, insulinase) was studied in adipose tissue and in liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats. Proteinase activity was found to be significantly decreased during diabetes and was restored to near normal levels in both tissues following insulin treatment. The insulin-mediated increase of proteinase activity in both tissues was partially or completely blocked by actinomycin D (an inhibitor of RNA synthesis) and by cyclohexamide (an inhibitor of protein synthesis). Kinetic analysis showed that the changes in proteinase activity of both liver and adipose tissues were accompanied by a change in Vmax (i.e., maximal enzyme activity) without a change in Km (i.e., substrate affinity). These data indicate that insulin functions as an inducer for neutral cysteine proteinase in both tissues. These alterations in the proteinase activity paralleled the alterations in the activity of a second insulin-degrading enzyme, glutathione-insulin transhydrogenase in adipose tissue (this paper) and in liver (previously published papers) under the same physiological conditions.     

枯草芽孢杆菌fmbJ产生的新型抗微生物物质体外抗NDV和IBDV的活性分析

测定了枯草芽孢杆菌fmbJ株产生的新型抗微生物物质的体外抗新城疫病毒(Newcastle disease virus,NDV)lasota株、传染性法式囊病病毒(Infectious Bursal Disease Virus,IBDV)哈尔滨(H)株作用。结果表明该新型抗微生物物质对鸡胚成纤维(Chicken Embryo Fibroblasts,CEF)细胞的TD50和TD0分别为128.95mg/L、25.79mg/L;对NDVlasota株、IBDV H株所致细胞病变效应有明显的抑制作用,可使细胞存活率显著升高;该抗微生物物质具有抗NDVlasota株、IBDV H株作用;并具有预防其感染及抑制其复制的作用。其抗病毒作用效果和病毒唑相当,由于其对CEF细胞的毒性较弱,可作为一种抗病毒药物进行开发研究。     

Proteinases and proteinase inhibitors as modulators of animal cell growth.

1. Three distinct lines of evidence indicate that proteinases are involved in the growth of cultured animal cells. 2. Endogenous growth-related proteinases have been identified, and exogenous proteinases can also stimulate cell proliferation, probably by different mechanisms. In some cases, higher concentrations of proteinases are cytotoxic. 3. Proteinase inhibitors, not surprisingly, inhibit cell growth, but can also be mitogenic at sub-inhibitory concentrations. 4. There must, therefore, be at least three major cellular processes in which proteinases or proteinase inhibitors can operate to exert a direct effect on cell proliferation. 5. Details of one action of an exogenous proteinase, typified by thrombin and the thrombin receptor, are becoming clear at the molecular level, but thrombin probably activates at least two intracellular signalling systems, as well as acting as a growth inhibitor in some situations. 6. Much remains to be investigated in other examples.     

Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes

An alkaline proteinase, previously identified in rat liver and heart, has been purified from the soluble fraction of human erythrocytes. The proteinase has an apparent molecular weight of 600 000 and is composed of eight subunits with molecular weights ranging from 32 000 to 21 000. The proteinase degrades both protein and synthetic peptide substrates with a broad pH optimum of 7.5-11.0. Among the synthetic peptides tested, tripeptides with arginine at the P1 position (e.g. Z-Val-Leu-Arg-4-methoxy-2-napthylamine and Boc-Leu-Gly-Arg-4-methylcoumarin-7-amide) are particularly good substrates. The proteinase appears to be sulfhydryl-dependent and is inhibited completely by mersalyl acid and by hemin; inhibitors of serine and metallo-type proteinases have no effect on proteinase activity. Interestingly, a variety of other proteinase inhibitors such as leupeptin, chymostatin and N-ethylmaleimide failed to completely inhibit protein-hydrolyzing activities of the enzyme. These results indicate that these activities may be accounted for by at least two different catalytic sites. Proteinase activity is stable in the presence of 1 M urea, 0.5% Triton X-100 or 0.03% SDS and is not affected by ATP. Based on the high molecular weight and sulfhydryl-dependence, we have named this proteinase macropain.