(1/485) Kallidin- and bradykinin-degrading pathways in human heart: degradation of kallidin by aminopeptidase M-like activity and bradykinin by neutral endopeptidase.

BACKGROUND: Since kinins kallidin (KD) and bradykinin (BK) appear to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of kinin-degrading enzymes should potentiate such effects. Indeed, it is believed that this mechanism is partly responsible for the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors. In the heart, enzymes other than ACE may contribute to local degradation of kinins. The purpose of this study was to investigate which enzymes are responsible for the degradation of KD and BK in human heart tissue. METHODS AND RESULTS: Cardiac membranes were prepared from the left ventricles of normal (n=5) and failing (n=10) hearts. The patients had end-stage congestive heart failure as the result of coronary heart disease or idiopathic dilated cardiomyopathy. Heart tissue was incubated with KD or BK in the presence or absence of enzyme inhibitors. We found no difference in the enzymes responsible for kinin metabolism or their activities between normal and failing hearts. Thus KD was mostly converted into BK by the aminopeptidase M-like activity. When BK was used as substrate, it was converted into an inactive metabolite BK-(1-7) mostly (80% to 90%) by the neutral endopeptidase (NEP) activity, with ACE unexpectedly playing only a minor role. The low enzymatic activity of ACE in the cardiac membranes, compared with that of NEP, was not due to chronic ACE inhibitor therapy, because the cardiac ACE activities of patients, whether receiving ACE inhibitors or not, and of normal subjects were all equal. CONCLUSIONS: The present in vitro study shows that in human cardiac membranes, the most critical step in kinin metabolism, that is, inactivation of BK, appears to be mediated mostly by NEP. This observation suggests a role for NEP in the local control of BK concentration in heart tissue. Thus inhibition of cardiac NEP activity could be cardioprotective by elevating the local concentration of BK in the heart.  (+info)

(2/485) Immunophenotype of myeloid cells in myelodysplastic syndromes and its clinical implications.

OBJECTIVE: To explore the immunophenotype of myeloid cells in myelodysplastic syndyomes (MDS) and its clinical implications. METHODS: A panel of monoclonal antibody was used to detect CD13+, CD33+, CD15+ and CD14+ antigens on the membrane surfaces of myeloid cells in the bone marrow from 51 MDS, 21 aplastic anemia (AA), 21 paroxysmal nocturnal hemoglobinuria (PNH) patients. 10 acute myeloblastic leukemia (AML) patients and 15 normal subjects by immunoenzymatic assay. The morphology and chromosome karyotype of bone marrow cells of MDS patients were also examined. RESULTS: CD14+, CD13+ and CD33+ cells in the bone marrow were more in MDS patients than in normal controls, AA patients and PNH patients. CD15+ cells in the bone marrow were less in MDS patients than in normal controls. CONCLUSIONS: The percentages of CD14+, CD13+ and CD33+ positive cells in the bone marrow of MDS patients were related to the percentage of myeloblasts, the chromosomal aberrations and the response to treatment. It indicated that there is immunophenotypic misexpression of myeloid cells in MDS patients. Immunophenotype analysis of myeloid cells might be useful for the diagnosis and treatment of MDS patients.  (+info)

(3/485) The flow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements.

BACKGROUND AND OBJECTIVE: Rapid identification of AML patients carrying the t(15;17) translocation for treatment decision-making is currently made on the basis of morphologic screening. However, the existence of both false positives and negatives highlights the need for more objective methods of screening AML cases and further molecular confirmation of the t(15;17) translocation. DESIGN AND METHODS: In the present study we analyzed a total of 111 AML cases in order to investigate whether immunophenotyping based on the assessment of multiple-stainings analyzed at flow cytometry could improve the sensitivity and specificity of morphologic identification of acute promyelocytic leukemia (APL) carrying the t(15;17) translocation. FISH analysis was used as a complementary technique for cases in which morphology and molecular biology yielded discrepant results. RESULTS: Concordant results between morphology and RT-PCR were found in 102/111 (91.8%) cases: 34 patients had M3/PML-RARalpha+ and 68 non-M3/PML-RARalpha- disease. Nine cases showed discrepants results. Multivariate analysis showed that the best combination of immunologic markers for discriminating between M3/PML-RARalpha+ and non-M3/PML-RARalpha- cases was that of the presence of heterogeneous expression of CD13, the existence of a single major blast cell population, and a characteristic CD34/CD15 phenotypic pattern (p<0.02). A score system based on these parameters was designed, and the 34 M3/PML-RARalpha+ cases showed a score of 3 (presence of the 3 phenotypic characteristics). In contrast, only 1 out of the 68 (1.3%) non-M3/PML-RARalpha- cases had this score, most o these latter cases (53/68, 78%) scoring either 0 or 1. Therefore, among these cases, immunophenotyping showed a sensitivity of 100% and a specificity of 99% for predicting PML/RARalpha gene rearrangements. Of the 9 cases in which morphology and molecular biology results were discrepant, four cases displayed M3 morphology without PML/RARalpha rearrangements by RT-PCR. In only one of these 4 cases did the immunophenotype score 3, this being the only FISH positive case. From the remaining five discrepant cases (non-M3 morphology while positive for PML/RARalpha) two cases had a phenotypic score of 3 and were FISH positive while the other three were negative by FISH. Upon repeating RT-PCR studies, two of these latter three cases became negative. INTERPRETATION AND CONCLUSIONS: Our results show that immunophenotyping may be of great value for quick screening of APL with PML/RARalpha rearrangements.  (+info)

(4/485) Dissociation between growth arrest and differentiation in Caco-2 subclone expressing high levels of sucrase.

Growth arrest and cell differentiation are generally considered temporally and functionally linked phenomena in small intestinal crypt cells and colon tumor cell lines (Caco-2, HT-29). We have derived a Caco-2 subclone (NGI3) that deviates from such a paradigm. In striking contrast with the parental cells, proliferative and subconfluent NGI3 cells were found to express sucrase-isomaltase (SI) mRNA and to synthesize relatively high levels of SI, dipeptidyl peptidase IV, and aminopeptidase N (APN). In postconfluent cells, little difference was seen in SI mRNA levels between Caco-2 and NGI3 cells, but the latter still expressed much higher levels of SI that could be attributed to higher rates of translation. APN expression was also greatly enhanced in NGI3 cells. To determine whether high levels of brush-border enzymes correlated with expression of cell-cycle regulatory proteins, we investigated their relative cellular levels in growing and growth-arrested cells. The results showed that the cyclin-dependent kinase inhibitors (p21 and p27) and D-type cyclins (D1 and D3) were all induced in postconfluent cells, but NGI3 cells expressed much higher levels of p21. This study demonstrated that cell growth and expression of differentiated traits are not mutually exclusive in intestinal epithelial cells and provided evidence indicating that posttranscriptional events play an important role in regulation of SI expression.  (+info)

(5/485) Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV.

The temporal association between O-glycosylation and processing of N-linked glycans in the Golgi apparatus as well as the implication of these events in the polarized sorting of three brush border proteins has been the subject of the current investigation. O-Glycosylation of pro-sucrase-isomaltase (pro-SI), aminopeptidase N (ApN), and dipeptidyl peptidase IV (DPPIV) is drastically reduced when processing of the mannose-rich N-linked glycans is blocked by deoxymannojirimycin, an inhibitor of the Golgi-located mannosidase I. By contrast, O-glycosylation is not affected in the presence of swainsonine, an inhibitor of Golgi mannosidase II. The results indicate that removal of the outermost mannose residues by mannosidase I from the mannose-rich N-linked glycans is required before O-glycosylation can ensue. On the other hand, subsequent mannose residues in the core chain impose no sterical constraints on the progression of O-glycosylation. Reduction or modification of N- and O-glycosylation do not affect the transport of pro-SI, ApN, or DPPIV to the cell surface per se. However, the polarized sorting of two of these proteins, pro-SI and DPPIV, to the apical membrane is substantially altered when O-glycans are not completely processed, while the sorting of ApN is not affected. The processing of N-linked glycans, on the other hand, has no influence on sorting of all three proteins. The results indicate that O-linked carbohydrates are at least a part of the sorting mechanism of pro-SI and DPPIV. The sorting of ApN implicates neither O-linked nor N-linked glycans and is driven most likely by carbohydrate-independent mechanisms.  (+info)

(6/485) Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A.

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.  (+info)

(7/485) Quantification of conversion and degradation of circulating angiotensin in rats.

The aim of the present study was to quantify with a uniform technique the rates of conversion of ANG I to ANG II in the lung and kidney and the degradation of both peptides to biologically inactive products in the pulmonary, renal, and systemic circulation. We infused the peptides intravenously, into the left ventricle, and into the left renal artery of rats and compared their effects on renal blood flow. The measured change in renal blood flow was used as a bioassay parameter to estimate the concentration of circulating ANG II. Mathematical analysis of our data allowed us to calculate conversion and degradation rates. Furthermore, the role of aminopeptidases A (EC and N (EC in the degradation of the peptides in the kidney was investigated by intrarenal infusion of the inhibitor amastatin. Our results show that the conversion rate of ANG I is 75% in the pulmonary and 21% in the renal circulation. Both peptides are degraded by 5% in the pulmonary, by 67% in the systemic, and by 93% in the renal circulation. Amastatin prevented 60% of the renal degradation of the peptides to inactive products, and this effect could be attributed to inhibition of aminopeptidase N. The results indicate that the converting capacity of the kidney is of minor importance for endocrine generation of ANG II but could be useful for the paracrine production.  (+info)

(8/485) Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13).

Human mast cells are known to arise from a CD34(+)/c-kit(+) progenitor cell population that also gives rise to neutrophils, eosinophils, basophils, and monocytes. To further characterize cells within the CD34(+)/c-kit(+) population that yield mast cells, this progenitor was additionally sorted for CD13, a myeloid marker known to appear early on rodent mast cells and cultured human mast cells, but not expressed or expressed at low levels on human tissue mast cells; and cultured in recombinant human (rh) stem cell factor (rhSCF), rh interleukin-3 (rhIL-3; first week only), and rhIL-6. Initial sorts revealed that although the majority of cells in culture arose from the CD34(+)/c-kit(+)/CD13(-) cell population, mast cells arose from a CD34(+)/c-kit(+)/CD13(+) progenitor cell that also gave rise to a population of monocytes. Sequential sorting confirmed that CD34(+)/c-kit(+)/CD13(+) cells in CD34(+)/c-kit(+)/CD13(-) sorts gave rise to the few mast cells observed in CD13(-) sorted cells. CD34(+)/c-kit(+)/CD13(+) cells plated as single cells in the presence of various cytokine combinations gave rise to pure mast cell, monocyte, or mixed mast cell/monocyte progeny. Addition of either rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or rhIL-5 to the CD34(+)/c-kit(+)/CD13(+) progenitor cell population cultured in rhSCF, rhIL-3, and rhIL-6 did increase the number of total cells cultured and in the case of rhIL-5, did increase total mast cell numbers. Neither rhGM-CSF or rhIL-5 led to additional cell populations, ie, even with the addition of rhGM-CSF or rhIL-5, only mast cells and monocytes grew from CD34(+)/c-kit(+)/CD13(+) cells. Thus, human mast cells and a population of monocytes arise from precursor cells that express CD34, c-kit, and CD13; and within which, are mast cell, monocyte, and mast/monocyte (bipotential) precursors.  (+info)