Two-photon fluorogenic probe for visualizing PGP-1 activity in inflammatory tissues and serum from patients.

A PGP-1-specific one/two-photon fluorogenic probe (BH1), capable of high sensitivity, super selectivity, and visual imaging of endogenous PGP-1 activity from live mammalian cells and serum/skin tissues from patients by using one/two-photon fluorescence microscopy (O/TPFM).

Neoadjuvant chemotherapy modifies serum pyrrolidone carboxypeptidase specific activity in women with breast cancer and influences circulating levels of GnRH and gonadotropins.

PURPOSE: Functional studies have demonstrated that gonadotropin-releasing hormone (GnRH) regulates cell proliferation, apoptosis, and tissue remodeling. GnRH is metabolized by the proteolytic regulatory enzyme pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3), which is an omega peptidase widely distributed in fluids and tissues. We previously reported a decrease in both rat and human Pcp activity in breast cancer, suggesting that GnRH may be an important local hormonal factor in the pathogenesis of breast cancer. Recently, we have described that postmenopausal women with breast cancer show lower levels of serum Pcp activity than control postmenopausal women. To determine the effect of neoadjuvant chemotherapy (NACT) on serum Pcp specific activity and circulating levels of GnRH, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and steroid hormones 17-ß-estradiol and progesterone in pre- and postmenopausal women diagnosed with infiltrating ductal carcinoma. METHODS: Serum Pcp activity was measured fluorometrically using pyroglutamyl-ß-naphthylamide. Circulating GnRH levels were dosed using a commercial RIA kit. Circulating LH and FSH levels were measured by enzyme immunoassays. Levels of steroid hormones were measured in serum samples by dissociation-enhanced lanthanide fluorescence immunoassay. RESULTS AND CONCLUSION: Our results show the effect of NACT on the hypothalamic-pituitary axis, with the consequent alteration of circulating gonadotropins in premenopausal women with breast cancer. However, the results obtained in postmenopausal women with breast cancer treated with NACT, that is, the significant decrease in the concentration of GnRH and FSH compared to control postmenopausal women, differ from those obtained for premenopausal women. The only difference between pre- and postmenopausal women is their hormonal profile at the beginning of the study, that is, the presence of menopause and the consequent alteration of the hypothalamic-pituitary-gonadal axis.

Serum pyroglutamyl aminopeptidase activity: a promising novel biomarker candidate for liver cirrhosis.

OBJECTIVE: As a reflect of tissue damage, serum aminopeptidases have been proposed as biomarkers of various diseases. In order to search new serologic markers for liver cirrhosis we conducted a preliminary study in which we analyzed a broad range of aminopeptidase activities in serum of controls and patients diagnosed with pancreatitis, hepatitis, and liver cirrhosis without distinction among the etiological type or the degree of severity of each condition. METHODS: Alanyl-, arginyl-, glutamyl-, cystinyl- pyroglutamyl-, and aspartyl-aminopeptidase activities were analyzed fluorometrically, using aminoacyl-ß-naphthylamides as substrates. In addition, various parameters, such as alanine transaminase, aspartate transaminase, alkaline phosphatase, total bilirubin, direct bilirubin, and gamma glutamyl transpeptidase were assayed as routine laboratory test for liver function. RESULTS: Compared with control group, alanyl- and arginyl-aminopeptidase activities increased nonspecifically in pancreatitis, hepatitis and liver cirrhosis, glutamyl- and cystinyl-aminopeptidases did not differ between groups and pyroglutamyl-aminopeptidase demonstrated that while pancreatitis and hepatitis did not differ between them and with controls, this activity decreased selectively in liver cirrhosis compared with all the rest of groups (p<0.001 vs. control and p<0.01 vs. pancreatitis and hepatitis). Aspartyl-aminopeptidase also decreased significantly (p<0.05) in liver cirrhosis compared with controls. Routine parameters for liver function test increased, as expected, in the three pathologies analyzed. CONCLUSIONS: Despite the heterogeneous composition of the three patient groups, the specific reduction of the levels of pyroglutamyl-aminopeptidase activity in serum of liver cirrhosis patients might be considered as a potential candidate to be included in a combination of markers for the diagnosis of this disease.

Putative relationship between hormonal status and serum pyrrolidone carboxypeptidase activity in pre- and post- menopausal women with breast cancer.

In breast cancer, hormonal changes are rather constant in post-menopausal women since they tend to vary only over long time spans. However, in pre-menopausal women, the development of breast cancer is associated with hormonal physiological variations. The aim of the present work was to analyse the changes in circulating levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in pre- and post-menopausal women that were healthy or with breast cancer, and their connection to serum pyrrolidone carboxypeptidase (Pcp) activity. We observed significant changes in the hormonal profile in post-menopausal women with breast cancer compared to the control group. In pre-menopausal women, we found significant changes in circulating GnRH levels with respect to the healthy group. Our present results support the existence of neuroendocrine misregulation that could be involved in tumour progression, with Pcp being a potentially new pharmacological target in breast cancer treatments.

Serum pyrrolidone carboxypeptidase activity in N-methyl-nitrosourea induced rat breast cancer.

Pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3) is an omega peptidase widely distributed in animal fluids and tissues and hydrolyses N-terminal pyroglutamic residues from biologically active peptides such as gonadotropin releasing hormone (GnRH). Previous results obtained by us showed a decrease in human breast cancer Pcp activity, suggesting that this enzyme activity or its putative substrates may play a major role in breast cancer pathogenesis. The aim of the present work is to analyse serum Pcp activity in N-methyl-nitrosourea (NMU) induced rat mammary tumours using pyroglutamyl-beta-naphthylamide as substrate. Serum Pcp activity was significantly lower in NMU-treated rats than in controls. Moreover, multiple regression analysis showed a significant correlation between Pcp activity and the number and size of tumours and the body weight of the animals. Since NMU-induced carcinomas are mainly oestrogen-dependent, the decrease observed in Pcp activity may reflect an increase in circulating levels of GnRH that lead to an increase in gonadal steroid hormones production responsible, at least in part, for the initiation and promotion of the disease.

Influence of dietary supplementation with olive oil on pyroglutamyl-beta-naphthylamide hydrolysing activity in serum and different tissues of mice.

Pyroglutamyl aminopeptidase is an omega peptidase which removes pyroglutamyl N-terminal residues from peptides and arylamide derivatives. To date, three distinct types of this enzyme have been described and called serum thyroliberinase, cytosolic pyroglutamyl aminopeptidase type I and membrane-bound pyroglutamyl aminopeptidase type II. The activity of all of them is thought to be involved in the regulation, more or less restricted in their substrate specificity, of various susceptible endogenous substrates such as TRH, GnRH, neurotensin, bombesin and anorexogenic peptide. It is well known that the type and amount of fat in the diet not only modify blood lipid concentrations, including cholesterol levels, but change the cell membrane lipid composition. Modifications in the composition and physical properties of the membrane lead to alterations in the activities of membrane-bound enzymes and carriers. The aim of this work was to compare the effect of a standard diet and a high fat diet (olive oil, 20% wt/wt) on pyroglutamyl-beta-naphthylamide hydrolysing activity, in serum and in soluble and membrane-bound fractions from different tissues of male mice. After ten weeks of feeding, pyroglutamyl-beta-naphthylamide hydrolysing activity was measured fluorometrically using pyroglutamyl-beta-naphthylamide as substrate. Mice fed the high fat diet had higher rates of body weight than controls starting from the second week of feeding. Serum total cholesterol concentrations were higher after feeding the high fat diet than after feeding the control diet. In serum, no changes were observed in the high fat group. In selected tissues, only pyroglutamyl-beta-naphthylamide hydrolysing activity was modified significantly in the soluble fraction, but not in the membrane-bound one, decreasing in the adrenal gland of high fat fed animals. The results may reflect functional modifications in susceptible endogenous substrates.

Prodrugs of peptides. 19. Protection of the pyroglutamyl residue against pyroglutamyl aminopeptidase by N-acyloxymethylation and other means.

The N-terminal pyroglutamyl group in several peptides is specifically cleaved by pyroglutamyl aminopeptidase (PAPase I). With the aim of protecting this group against enzymatic cleavage by the prodrug approach, various derivatives of L-pyroglutamyl benzylamide, used as a PAPase I sensitive model pyroglutamyl peptide, were prepared and their stability characteristics determined. The derivatives studied included phenoxycarbonyl, phthalidyl, hydroxymethyl and actoxymethyl derivatives, all formed at the pyroglutamyl NH-moiety. Whereas L-pyroglutamyl benzylamide was rapidly hydrolyzed by PAPase I, all the derivatives were resistant to cleavage by the enzyme. On the other hand, these derivatives, with the exception of the N-phenoxycarbonyl derivative, were readily converted to the parent pyroglutamyl benzylamide by spontaneous or plasma catalyzed hydrolysis, the half-lives of conversion in 80% human plasma being in the range 2.3-8.4 h. The major degradation reaction of the N-phenoxycarbonyl derivative in both buffer and plasma solutions was hydrolytic opening of the pyrrolidone ring. The pH-rate profiles for the degradation of the compounds in aqueous solution were obtained and both specific acid and base catalytic reactions as well as a spontaneous reaction were observed. The results suggest that N-phthalidylation, N-hydroxymethylation and N-acyloxymethylation of pyroglutamyl peptides may be useful prodrug approaches to protect such peptides against cleavage by pyroglutamyl aminopeptidase and hence to improve their delivery characteristics.

Thyrotropin-releasing hormone-degrading enzyme in human serum is classified as type II of pyroglutamyl aminopeptidase: influence of thyroid status.

The characteristics of thyrotropin-releasing hormone (TRH)-degrading enzyme in human serum were studied. Serum was incubated in 0.1 M phosphate buffer containing [proline-3H]TRH at 37 degrees C. A thin layer chromatography analysis of TRH degradation did not show any radioactive peak located in an acid TRH position, but apparent radioactive peaks corresponding to His-Pro and His-ProNH2 occurred in the presence of p-hydroxymercuriphenyl sulfonic acid, an inhibitor of proline dipeptidase. With ion exchange paper chromatography, the formation of 3H-labeled His-Pro and His-ProNH2 was estimated as an end point in the measurement of pyroglutamyl aminopeptidase (pGlu-peptidase) activity. An assay using p-hydroxymercuriphenyl sulfonic acid was developed to sensitively quantitate the pGlu-peptidase. Neither bacitracin nor p-chloromercuribenzoic acid increased the activity of pGlu-peptidase. The addition of EDTA, dithiothreitol, and o-phenanthroline significantly inhibited pGlu-peptidase activity, but neither iodoacetamide nor ethylmaleimide altered its activity. The pGlu-peptidase had a stereotypic specificity for the tripeptide, pGlu-His-ProNH2 of TRH, and its Km was 44.9 microM. The pGlu-peptidase activity was not changed by either hyper- or hypothyroidism. The present data indicate that a TRH-degrading enzyme in human serum possesses a nature identical to type II of pGlu-peptidase which is not altered by thyroid status.

Thyroid status influences rat serum but not brain TRH pyroglutamyl aminopeptidase activities.

Serum and brain cytosol contains pyroglutamyl aminopeptidase activity that converts TRH to His-ProNH2 (TRH PAPase). Whereas serum TRH PAPase has specificity for TRH, this is not the case for brain cytosol PAPase. Recent reports indicate that a brain membrane fraction contains TRH PAPase that is specific for TRH and has a remarkable similarity to serum TRH PAPase. In the present studies, a method for measuring serum TRH PAPase activity and the activities of the membrane and cytosol brain TRH PAPase enzymes are described. The effect of thyroid status on these different TRH PAPase activities was determined. In hypothyroid rats serum TRH PAPase activity was decreased, whereas in rats treated with supraphysiological doses of T4 it was increased. In contrast, the cytosolic and the membrane TRH PAPase enzymes in brain were not affected by thyroid status. It is concluded that the membrane-associated brain TRH PAPase differs from the serum TRH PAPase in terms of its response to thyroid hormone. In addition, the previously reported effects of thyroid status on rat serum TRH degrading activity are explained by the finding that thyroid hormone increases serum TRH PAPase activity.