phosphoramidon and pepstatin

To design a releasable PEGylated TNF-α (rPEG-TNF-α), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF-α (PEG-TNF-α), facilitating its clinical use for anti-tumor therapy. Comparative pharmacokinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ∼60-fold greater than that of unmodified TNF-α. In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9-fold more potent, respectively, than PEG-TNF-α. Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α.

Topics: Animals; Antineoplastic Agents; Cathepsin B; Cell Line, Tumor; Cell Survival; Dipeptides; Dose-Response Relationship, Drug; Drug Compounding; Drug Evaluation, Preclinical; Glycopeptides; Humans; Hydrogen-Ion Concentration; Leucine; Lysosomes; Male; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Pepstatins; Polyethylene Glycols; Protease Inhibitors; Rats; Time Factors; Tumor Burden; Tumor Necrosis Factor-alpha

Matrix metalloproteinases (MMPs) are important in dentinal caries, and analysis of recent data demonstrates the presence of other collagen-degrading enzymes, cysteine cathepsins, in human dentin. This study aimed to examine the presence, source, and activity of cysteine cathepsins in human caries. Cathepsin B was detected with immunostaining. Saliva and dentin cysteine cathepsin and MMP activities on caries lesions were analyzed spectrofluorometrically. Immunostaining demonstrated stronger cathepsins B in carious than in healthy dentin. In carious dentin, cysteine cathepsin activity increased with increasing depth and age in chronic lesions, but decreased with age in active lesions. MMP activity decreased with age in both active and chronic lesions. Salivary MMP activities were higher in patients with active than chronic lesions and with increasing lesion depth, while cysteine cathepsin activities showed no differences. The results indicate that, along with MMPs, cysteine cathepsins are important, especially in active and deep caries.

Topics: Adolescent; Adult; Age Factors; Cathepsin B; Cathepsins; Child; Cysteine Proteases; Cysteine Proteinase Inhibitors; Dental Caries; Dental Pulp Exposure; Dentin; Fluorescent Dyes; Glycopeptides; Humans; Leucine; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Metalloendopeptidases; Middle Aged; Odontoblasts; Oligopeptides; Pepstatins; Protease Inhibitors; Saliva; Serine Proteinase Inhibitors; Spectrometry, Fluorescence; Young Adult

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glycopeptides; Losartan; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Pepstatins; Peptides, Cyclic; Protease Inhibitors; Protein Precursors; Rats; Rats, Wistar; Renin

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.

Topics: Animals; Binding Sites; Binding, Competitive; Cell Adhesion; Cell Division; Culture Media; Glycopeptides; Humans; Hyaluronan Receptors; Hyaluronic Acid; Melanoma; Melanoma, Experimental; Metalloendopeptidases; Mice; Mice, Nude; Neoplasm Proteins; Pepstatins; Phenanthrolines; Protease Inhibitors; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Deletion; Sulfones; Transfection; Tumor Cells, Cultured

Most early-onset forms of Alzheimer's disease are due to missense mutations located on two homologous proteins named presenilin 1 and 2 (PS1 and PS2). Several lines of evidence indicate that PS1 and PS2 undergo various post-transcriptional events including endoproteolytic cleavages, giving rise to 28-30 kD N-terminal (NTF) and 18-20 kD C-terminal (CTF) fragments that accumulate in vivo. Whether the biological activity of presenilins is borne by the processed fragments or their holoprotein precursor remains in question. We have examined the putative control of beta APP maturation by CTF-PS1/PS2 and the catabolic process of the latter proteins by the multicatalytic complex, proteasome.. We transiently and stably transfected HEK293 cells with CTF-PS1 or CTF-PS2 cDNA. We examined these transfectants for their production of A beta 40, A beta 42, and APP alpha by immunoprecipitation using specific polyclonals. The effect of a series of proteases inhibitors on the immunoreactivity of CTF-PS1/PS2 was examined by Western blot. Finally, the influence of proteasome inhibitors on the generation of beta APP fragments by CTF-expressing cells was assessed by combined immunoprecipitation and densitometric analyses.. We showed that transient and stable transfection of CTF-PS1 and CTF-PS2 cDNAs in human cells leads to increased secretion of APP alpha and A beta, the maturation products of beta APP. Furthermore, we demonstrated that two proteasome inhibitors, lactacystin and Z-IE(Ot-Bu)A-Leucinal, prevent the degradation of both CTFs. Accordingly, we established that proteasome inhibitors drastically potentiate the phenotypic increased production of APP alpha and A beta elicited by CTF-PS1/PS2.. Our data establish that the C-terminal products of PS1 and PS2 maturation exhibit biological activity and in particular control beta APP maturation upstream to alpha-and beta/gamma-secretase cleavages. This function is directly controlled by the proteasome that modulates the intracellular concentration of CTFs.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Glycopeptides; Humans; Leucine; Leupeptins; Membrane Proteins; Multienzyme Complexes; Oligopeptides; Pepstatins; Presenilin-1; Presenilin-2; Proteasome Endopeptidase Complex; Recombinant Proteins; Sulfones; Transfection

1. It is generally accepted that endothelial cells secrete endothelin (ET) to the underlying media which mediates the contractile effects of ET. However, there is some evidence that animal vascular smooth muscle cells (VSMCs) also secrete ET. We cultured VSMCs from human vessels representative of a number of different vascular beds to determine whether human VSMCs endogenously secrete ET. 2. VSMCs explanted from adult arterial vessels secrete picomolar quantities of immunoreactive mature ET: coronary artery 226.6 +/- 58.8 pM/10(6) cells (n = 7), thoracic aorta 169.5 +/- 105.4 pM/10(6) cells (n = 3), left internal mammary artery 102.4 +/- 23.1 pM/10(6) cells (n = 3) and saphenous vein 69.4 +/- 19.9 pM/10(6) cells (n = 3), as well as from umbilical vein (HUVSMCs) 38.3 +/- 4.3 pM/10(6) cells (n = 3). Secretion of immunoreactive big ET-1 was also detected: coronary artery 249.1 +/- 59.4 pM/10(6) cells (n = 7), thoracic aorta 120.0 +/- 13.4 pM/10(6) cells (n = 3), left internal mammary artery 170.0 +/- 68.2 pM/10(6) cells (n = 3), saphenous vein 105.1 +/- 30.7 pM/10(6) cells (n = 3) and from umbilical vein 146.3 +/- 7.4 pM/10(6) cells (n = 3). Comparable, intracellular levels of immunoreactive big ET-1 and mature ET were also detected in cultured VSMCs. 3. Since enzyme-dispersed VSMCs are thought to be more differentiated and more closely resemble their in vivo counterparts, and these enzyme-dispersed VSMCs from human umbilical vein (HUVSMCs) also secreted the greatest levels of immunoreactive peptides, they were characterized further. Reverse transcription-polymerase chain reaction assay demonstrated that HUVSMCs express ET-1 mRNA. High performance liquid chromatography coupled to radioimmunoassay revealed that HUVSMCs secrete ET-1 and ET-3, in addition to big ET-1. However, levels of ET are not altered by 100 AM phosphoramidon,an inhibitor of metalloproteases or by 100 microM pepstatin A, an aspartyl protease inhibitor.4. In concordance, KD and Bmax values for [125I]-ET-l saturation binding are not altered in HUVSMC cultures incubated for 24 h with 100 microM phosphoramidon (431 +/- 218 PM and 31.1 +/- 12.7 fmol mg-1;mean =/- s.e.mean, n = 3) or 100 microM pepstatin A (381 +/- 169PM and 19.9 +/- 7.8 fmol mg-1, n = 3) as compared to controls (355 +/- 99 pM and 33.3 +/- 9.3 fmol mg-1; n = 3). This observation indicates the absence of an autocrine 'unmasking' effect for ET receptors.5. HUVSMCs synthesize and secrete immunoreactive ET-1, ET-3 and big ET-1, and p

Topics: Antimalarials; Base Sequence; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Female; Glycopeptides; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Muscle, Smooth, Vascular; Pepstatins; Protease Inhibitors; Protein Precursors; Proteins; Radioimmunoassay; RNA, Messenger; Umbilical Veins

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and non-isotopic measurement of endothelin-1 and big endothelin-1. The sensitivities of detection were 0.5 and 0.3 fmol/well, with ED50 values of 13 and 12 fmol/well for the endothelin-1 and big endothelin-1 assays, respectively. Each assay is highly selective for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only recognizes the 21-amino acid biologically active peptide. 2. The two assays were used to measure the effects of two classes of protease inhibitor on the basal release of enothelin-1 and big endothelin-1 from cultured first-passage human umbilical vein endothelial cells. 3. The secretion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P < 0.05), with a concomitant increase in the secreted levels of big endothelin-1 (P < 0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significant decrease in the secretion of endothelin (P < 0.05). However, unlike phosphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affected the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antimalarials; Cells, Cultured; Endothelin-1; Endothelins; Endothelium; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Glycopeptides; Humans; Metalloendopeptidases; Pepstatins; Protein Precursors; Umbilical Veins

The modulation of endothelin (ET) release from endothelial cells was investigated as a function of cell density. The present study examined the release of ET from bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC) at various cell densities, as well as the effects of phosphoramidon, thiorphan and pepstatin on ET release at different densities. ET release from BPAEC and BAEC decreased as cell density increased. This cell density effect was not observed with prostacyclin release from either BPAEC or BAEC. Phosphoramidon (1 mM) inhibited ET release at every density examined for both BPAEC and BAEC. Thiorphan (1 mM) inhibited ET release from BPAEC weakly at low density and had no effect on ET release from BAEC. Pepstatin (1 mM) slightly inhibited ET release in BPAEC at the lowest density and had no effect at any other cell density for either cell type. These protease inhibitors had no effect on cell viability as determined by trypan blue exclusion and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion assay. This study supports the concept that ET release is modulated by endothelial cell density. In addition, these data demonstrate that phosphoramidon, which presumably inhibits the endothelin converting enzyme, can inhibit ET release over a range of cell densities without affecting cell viability.

Topics: Animals; Cattle; Cell Count; Cell Survival; Dose-Response Relationship, Drug; Endothelins; Endothelium, Vascular; Glycopeptides; Pepstatins; Thiorphan

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme in vascular smooth muscle. Like cultured endothelial cells, noncultured vascular smooth muscle homogenate of bovine carotid arteries, converts human big ET- 1 to ET-1 at pH 3.0, pH 5.0 and pH 7.0, and the apparent ratio of these three activities is about 6:5:1, respectively. Peptides generated during incubation of the homogenate and big ET- 1 at the three pHs were identified as ET- 1 by radioimmunoassay and high performance liquid chromatography. The two acid enzymes are in the cytosol (103,000xg sup) and are inhibited by pepstatin A, while the neutral enzyme is sensitive to EDTA or phosphoramidon; 73% of the neutral enzyme activity was membrane-bound and the remainder (27%) cytosolic.

Topics: Animals; Aspartic Acid Endopeptidases; Carotid Arteries; Cattle; Chromatography, High Pressure Liquid; Cytosol; Edetic Acid; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Isoenzymes; Kinetics; Metalloendopeptidases; Muscle, Smooth, Vascular; Pepstatins; Protease Inhibitors; Radioimmunoassay

Human polymorphonuclear leukocytes (PMNs) converted human big endothelin (bET; 2 microM) to an endothelin-1 (ET-1) like contractile factor, as assessed by bioassay. The generation of this ET-1 like activity was rapid (minutes), time-dependent and more pronounced in non-activated cells, suggesting a partial degradation by activated PMNs. Phosphoramidon (54 micrograms/ml) inhibited the formation of this contractile factor, whereas phenylmethylsulfonylfluoride (PMSF; 25 micrograms/ml), pepstatin A (1 microgram/ml) or epoxysuccinyl-L-leucylamido-(guanidino)butane (E-64; 10 micrograms/ml) did not. Incubations of activated PMNs with PMSF significantly potentiated the generation of ET-1 like activity and selectively inhibited the degradation of [125I]ET-1 by activated PMNs. These findings indicate that human PMNs contain and/or release neutral proteases, which can both rapidly produce and degrade ET-1, an observation which may have important (patho)physiologic implications.

Topics: Animals; Endothelins; Glycopeptides; Humans; In Vitro Techniques; Jugular Veins; Kinetics; Leucine; Muscle, Smooth, Vascular; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pepstatins; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Rabbits