Study of gherkin lactase in cell culture and in seedlings.

A synthetic substrate replacing lactose has facilitated application of a simple, rapid and sensitive method for the identification and determination of extracellular and intracellular gherkin lactase. The intracellular enzyme activity was estimated from the cell suspension, while the extracellular enzyme activity was established within the cell free cultivation medium. A suspension of gherkin cells was permeabilized by Tween 20, or Tween 80, or hexadecyltrimethyl ammonium bromide, or hexadecylpyridinium chloride or ethanol added one at a time and then immobilized by glutaraldehyde.

Role of dipeptidyl peptidase IV (DP IV)-like enzymes in T lymphocyte activation: investigations in DP IV/CD26-knockout mice.

Background: Dipeptidyl peptidase IV (DP IV, CD26) and DP IV-like enzymes, such as dipeptidyl peptidase II (DP II), dipeptidyl peptidase 8 (DP8), and dipeptidyl peptidase 9 (DP9), have been recognized to regulate T lymphocyte activation. Lys[Z(NO2)]-thiazolidide (LZNT) and Lys[Z(NO2)]-pyrrolidide (LZNP), non-selective inhibitors of DP IV-like activity known to target DP IV as well as DP II, DP8, and DP9, suppress T lymphocyte proliferation in vitro. Moreover, these inhibitors are capable of attenuating the severity of autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and experimental arthritis, a model of human rheumatoid arthritis, in vivo, particularly in combination with inhibitors of aminopeptidase N (APN, CD13) enzymatic activity.

Recent insights into the role of dipeptidyl aminopeptidase IV (DPIV) and aminopeptidase N (APN) families in immune functions.

Background: In the past, different research groups could show that treatment of immune cells with inhibitors of post-proline splitting dipeptidyl aminopeptidases leads to functional changes in the immune system consistent with immunosuppression. This is due to the inhibition of proliferation of lymphocytes and the production of inflammatory cytokines of the TH1, TH2, and TH17, cells as well as the induction of immunosuppressive cytokines, such as transforming growth factor-beta1 (TGF-beta1) and interleukin (IL)-1RA. Until recently, most of the effects of these inhibitors on immune functions were attributed to the inhibition of dipeptidyl aminopeptidase IV (DPIV/CD26).

Non-substrate peptides influencing dipeptidyl peptidase IV/CD26 activity and immune cell function.

Investigations using inhibitors of dipeptidyl peptidase IV (DP IV) activities and DP IV-/- mice indicated an immunoregulatory role of DP IV that could not be compensated by DP IV-like enzymes. The HIV-1 Tat protein was identified as the first natural inhibitor of DP IV and as immunosuppressor. This review summarizes our investigations on the identification of the amino acid motif of Tat responsible for DP IV inhibition and of endogenous DP IV-inhibitory ligands that suppress immune cell activation.

DP IV/CD26, APN/CD13 and related enzymes as regulators of T cell immunity: implications for experimental encephalomyelitis and multiple sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system. Peptidases like dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) play a regulatory role in T cell activation and represent potential targets for the treatment of inflammatory disorders. Synthetic inhibitors of DP IV and/or APN enzymatic activity induce production of the immunosuppressive cytokine TGF-beta1 and subsequently suppress DNA synthesis and Th1 cytokine production of activated human T cells.

Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters.

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2).

Inhibitors of dipeptidyl peptidase IV-like activity mediate antifibrotic effects in normal and keloid-derived skin fibroblasts.

Suppression of collagen and matrix synthesis and inhibition of the fibrogenic cytokine transforming growth factor-beta(1) (TGF-beta(1)) is a major therapeutic goal in the treatment of fibrosis and keloids. Inhibitors of dipeptidyl peptidase IV (DP IV)-like activity affect cell growth and cytokine production and are currently under investigation for the treatment of metabolic, autoimmune and inflammatory diseases. We show here that the inhibitors of DP IV-like activity, Lys[Z(NO(2))]-thiazolidide and Lys[Z(NO(2))]-pyrrolidide, suppress proliferation in human skin fibroblasts and keloid-derived skin fibroblasts in vitro.

Dual inhibition of dipeptidyl peptidase IV and aminopeptidase N suppresses inflammatory immune responses.

The ectopeptidases dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) are known to regulate T cell activation. Since selective inhibitors of DP IV and APN suppress DNA synthesis and cytokine production of stimulated T cells in a TGF-beta1-dependent manner, we tested whether combined application of DP IV and APN inhibitors enhances this immunomodulatory effect. The results show that simultaneous application of DP IV and APN inhibitors significantly suppressed DNA synthesis in mitogen- or anti-CD3-stimulated human T cells in vitro when compared to the use of a single DP IV or APN inhibitor.

Recognition of 2-aminothiazole-4-acetic acid derivatives by the peptide transporters PEPT1 and PEPT2.

The H(+)/peptide cotransporters PEPT1 and PEPT2 have gained considerable interest in pharmaceutical sciences as routes for drug delivery. It is, therefore, of interest to develop uncommon artificial substrates for the two carriers. This study was initiated to investigate the binding affinity of 2-aminothiazole-4-acetic acid (ATAA) conjugates with amino acids to PEPT1 and PEPT2.

TGF-beta1-mediated control of central nervous system inflammation and autoimmunity through the inhibitory receptor CD26.

The T cell marker CD26/dipeptidyl peptidase (DP) IV is associated with an effector phenotype and markedly elevated in the human CNS disorder multiple sclerosis. However, little is known about the in vivo role of CD26/DP IV in health and disease, and the underlying mechanism of its function in CNS inflammation. To directly address the role of CD26/DP IV in vivo, we examined Th1 immune responses and susceptibility to experimental autoimmune encephalomyelitis in CD26(-/-) mice.

Bioassay for the determination of the intra- and extracellular activity of aminopeptidases in immobilized tomato cells.

Permeabilized tomato cells were cross-linked with glutaraldehyde in the absence of a carrier. The immobilized cells demonstrated significantly lower aminopeptidase (AP) activities than untreated control cells. However, when immobilized with pectate and alginate gels, the tomato cells retained their AP activities.

Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation.

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors.

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13)--a novel approach for the treatment of inflammatory bowel disease.

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-beta1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD). The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis.

Attractin, a dipeptidyl peptidase IV/CD26-like enzyme, is expressed on human peripheral blood monocytes and potentially influences monocyte function.

The ectoenzyme dipeptidyl peptidase IV (DP IV; CD26) was shown to play a crucial role in T cell activation. Several compounds inhibiting DP IV-like activity are currently under investigation for the treatment of Type 2 diabetes, rheumatoid arthritis, colitis ulcerosa, psoriasis, multiple sclerosis, and other diseases. In the present study, we show that human peripheral blood monocytes express a DP IV-like enzyme activity, which could be inhibited completely by the synthetic DP IV inhibitor Lys[Z(NO(2))]-thiazolidide.

Structural requirements for the substrates of the H+/peptide cotransporter PEPT2 determined by three-dimensional quantitative structure-activity relationship analysis.

The renal type H(+)/peptide cotransporter PEPT2 has a substantial influence on the in vivo disposition of dipeptides and tripeptides as well as peptide-like drugs within the body, particularly in kidney, lung, and the brain. The comparative molecular similarity indices analysis (CoMSIA) method was applied to identify those regions in the substrate structures that are responsible for recognition and for differences in affinity. We have developed a comprehensive 3D quantitative structure-activity relationship (3D-QSAR) model based on 83 compounds that is able to explain and predict the binding affinities of new PEPT2 substrates.

Inhibitors of dipeptidyl peptidase IV and aminopeptidase N target major pathogenetic steps in acne initiation.

Acne is a chronic disease hallmarked by sebaceous hyperplasia, follicular hyperkeratosis, and inflammation. Parallel targeting of these factors is required to treat acne effectively. Inhibitors of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) show strong anti-inflammatory effects on immune cells and therapeutic efficacy in autoimmune disorders.

Serotonin, L-tryptophan, and tryptamine are effective inhibitors of the amino acid transport system PAT1.

The proton-coupled amino acid transporter PAT1, cloned recently from brain and intestine, mediates the uphill transport of l- and d-proline, l-alanine, glycine, taurine, d-serine, GABA, and many other related compounds and drugs. Here we describe the novel finding that l-tryptophan and its derivatives tryptamine, 5-hydroxy-l-tryptophan, serotonin, and indole-3-propionic acid strongly inhibit H+-dependent l-[3H]proline uptake into Caco-2 cells with inhibition constants (K(i)) of 0.9 to 6.

Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1: Synthesis and kinetic characterization as aminopeptidase P inhibitors.

Novel, potent inhibitors of aminopeptidase P, containing a 3-amino-2-hydroxy acid and a proline or a proline analogues, have been prepared. One part of the bestatin-derived inhibitors was found to inhibit APP from Escherichia coli and from rat intestine according to a mixed-type mechanism, with Ki values up to 1.26 microM.

Three-dimensional quantitative structure-activity relationship analyses of beta-lactam antibiotics and tripeptides as substrates of the mammalian H+/peptide cotransporter PEPT1.

The utilization of the membrane transport protein PEPT1 as a drug delivery system is a promising strategy to enhance the oral bioavailability of drugs. Since very little is known about the substrate binding site of PEPT1, computational methods are a meaningful tool to gain a more detailed insight into the structural requirements for substrates. Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using the comparative molecular similarity indices analysis (CoMSIA) method were performed on a training set of 98 compounds.

The influence of 1-aminocyclopentane-1-carboxylic acid at position 2 or 3 of AVP and its analogues on their pharmacological properties.

This study describes the synthesis and some pharmacological properties of eight new analogues of arginine vasopressin (AVP) substituted at position 2 or 3 with cycloleucine (1-aminocyclopentane-1-carboxylic acid, Apc). All new peptides were tested for their pressor, antidiuretic and uterotonic in vitro potency. The Apc3 modification resulted in an almost complete loss of potency in all three tests, which is interpreted as a loss of interaction with all three neurohypophyseal hormone receptors.

Analysis of the transport properties of side chain modified dipeptides at the mammalian peptide transporter PEPT1.

This study was initiated to examine systematically the effect of side chain modifications at dipeptides on their transport via PEPT1. We synthesized a series of Xaa(R)-Ala and Ala-Xaa(R) dipeptides with the functional groups of the side chains modified by structurally different blocking groups R. Recognition and transport of these derivatives by PEPT1 was measured in Caco-2 cells, in transgenic Pichia pastoris cells and in Xenopus laevis oocytes expressing PEPT1.

A continuous fluorimetric assay for aminopeptidase P detailed analysis of product inhibition.

Aminopeptidase P (APP; EC 3.4.11.