Metallothionein Biosynthesis in Human RBC Precursors

The in vitro biosynthesis of metallothionein (MT) has been investigated in RBC precursors from human cord blood in order to support the hypothesis for the nucleated precursor origin of MT in human red blood cells (RBC). Human RBC precursors are obtained by (i) separating glycophorin A+ (gly A+) cells using a magnetic cell sorting (MACS) technique and by (ii) ex vivo expansion of precursors BFU-E (burst forming unit-erythroid) on methylcellulose semi-solid culture media from mononuclear cells of cord blood. Biosynthesis of MT is detected at the protein level, by immuno-histochemical staining using a mouse monoclonal antibody (E9) in ex vivo expanded RBC precursors obtained from BFU-E. Expression of MT is also detected at the mRNA level by MT specific reverse transcriptase polymerase chain reaction (RT-PCR) both in ex vivo expanded precursors from BFU-E and in MACS separated gly A+ cells. In addition, the expression of the fetal form of MT, MT-0 (also known as MT-1H) at the mRNA level in glycophorin A+ cells, is also confirmed by cDNA sequencing. With these observations, to our knowledge, MT biosynthesis in human erythroid precursors is reported for the first time. Moreover, the current findings of MT-0 expression at the mRNA level in gly A+ RBC precursors of hCB has added one more member in the list of cells/organs like fetal liver, human monocytes, non-neoplastic tissues of adenocarcinoma etc., in which the expression of the human fetal form of MT, i.e. MT-0, has also been reported

Postgenomic characterization of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning proteins. Their importance in drug development has been proven over and over again. Therefore, they remain one of the most significant groups of molecules to be characterized. In the postgenomic era, the methods used for the characterization of GPCRs have dramatically changed: the predicted orphan receptors are now often used to ascertain the ligands (reverse pharmacology), whereas, in the past, the bioactive ligand was used to identify the receptor (classic approach). in this review, we will give an overview of the recent postgenomic functional assays that are frequently used to link the orphan GPCR of both vertebrate and invertebrate organisms with their ligands.status: publishe

Isolation of angiotensin converting enzyme from testes of Locusta migratoria (Orthoptera)

By means of a tracer assay using a labeled synthetic angiotensin converting enzyme (ACE) substrate hippurylglycylglycine, we have detected high ACE activity in the testes of the African migratory locust, Locusta migratoria. Lower, but significant, ACE activity was observed in midgut and hemolymph. In a two-step purification procedure involving anion exchange and gel permeation chromatography, we have purified LomACE from the locust testes. The enzyme of approximately 80 kDa shows substantial amino-acid sequence homology with ACE from both vertebrate and invertebrate origin. The ACE identity of the purified enzyme was further confirmed by cDNA cloning of the Locusta ACE fragment, which, after in silico translation, revealed a mature protein of 623 amino acids with a large structural similarity to other known ACE proteins.status: publishe

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K-m values of Neb-ODAIF and Neb-ODAIFI-9 (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 muM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K-m/i = 90 muM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans. (C) 2002 Elsevier Science Inc. All rights reserved.status: publishe

Immunocytochemical distribution of angiotensin-I converting enzyme in the central nervous system of insects and speculations about its possible function

Insect peptidyl-dipeptidase A [angiotensin I - converting enzyme (ACE)] is a soluble single-domain peptidyl-dipeptidase that has many properties in common with the C-domain of mammalian somatic ACE and with the single-domain mammalian ACE. In agreement with a variety of insects, immunocytochemical studies reveal the presence of an ACE-like protein in Locusta migratoria. ACE-like immunoreactivity is present in neurosecretory cells of the pars intercerebralis. These cells have axons projecting into the nervus corporis cardiaci I and into the storage part of the corpus cardiacum, a neuroendocrine organ directly releasing into the aorta. The localisation of ACE in neurosecretory cells is consistent with its proposed role as a processing enzyme that is involved in the generation of active peptide hormones.status: publishe

Characterization of four substrates emphasizes kinetic similarity between insect and human C-domain angiotensin-converting enzyme

Angiotensin converting enzyme (ACE) was already discovered in insects in 1994, but its physiological role is still enigmatic. We have addressed this problem by purifying four new ACE substrates from the ovaries of the grey fleshfly, Neobellieria bullata . Their primary structures were identified as NKLKPSQWISLSD (Neb -ODAIF- 1(1-13) ), NKLKPSQWI (Neb -ODAIF- 1(1-9) ), SLKPSNWLTPSE (Neb -ODAIF- 2) and LEQIYHL. Database analysis showed significant homology with amino acid sequence stretches as present in the N-terminal part of several fly yolk proteins. An antiserum raised against Neb -ODAIF-1(1-9) immunostained one out of three yolk protein bands of SDS/PAGE-separated fly haemolymph and egg homogenate, thus confirming that these peptides originate from a yolk protein gene product. Kinetic analysis of these peptides and of the peptides Neb -ODAIF and Neb -ODAIF- 1(1-7) with insect ACE and human ACE show both similar and unique properties for insect ACE as compared with human C-domain ACE.status: publishe

Captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfly Neobellieria bullata

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata, In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE, Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal-induced trypsin peak in the midgut and elevated levels of protein meal-induced yolk polypeptides in the hemolymph, The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. (C) 2001 Wiley-Liss, Inc.status: publishe

In vitro degradation of the Neb-Trypsin Modulating Oostatic Factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences HPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF. (C) 2001 Elsevier Science Ltd. All rights reserved.status: publishe

Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport

The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from similar to3 min in L. maderae to similar to 25 min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments Asn-Pro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph.status: publishe