Search

Recently, the interest in the plant proteases has greatly increased. However, only a few of proteases are isolated from the hugely produced oilseeds for the practical utilizations. In this study, the raw sesame milk prepared from peeled sesame seeds was separated into floating, skim, and precipitate fractions by centrifugation. The predominant aspartic endopeptidases and serine carboxypeptidases, which exerted high synergetic activity at pH 4.5-5 and 50-60 °C, were identified in the skim by the liquid chromatography tandem mass spectrometry, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protease inhibitor assay, trichloroacetic acid-nitrogen soluble index (TCA-NSI), and free amino acid analyses. By incubating the mixture (protein content, 2%) of skim and precipitate at pH 4.5 and 50 °C for 6 h, the TCA-NSI and free amino acids achieved to 38.42% and 3148 mg/L, respectively. Moreover, these proteases efficiently degraded the proteins from soybean, peanut, and bovine milk., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The changes in protease activities in embryonic axes during the first days of bean (Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos.

The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.

Leucine carboxypeptidase (EC 3.4.16) activity in Actinomucor elegans bran koji was investigated via absorbance at 507 nm after stained by Cd-nihydrin solution, with calibration curve A, which was made by a set of known concentration standard leucine, calibration B, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations inactive crude enzyme extract, and calibration C, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations crude enzyme extract. The results indicated that application of pure amino acid standard curve was not a suitable way to determine carboxypeptidase in complicate mixture, and it probably led to overestimated carboxypeptidase activity. It was found that addition of crude exact into pure amino acid standard curve had a significant difference from pure amino acid standard curve method (p < 0.05). There was no significant enzyme activity difference (p > 0.05) between addition of active crude exact and addition of inactive crude kind, when the proper dilute multiple was used. It was concluded that the addition of crude enzyme extract to the calibration was needed to eliminate the interference of free amino acids and related compounds presented in crude enzyme extract.

Highly selective luminescent probes, QLUC-TYR and LUC-GLU, for detection of carboxypeptidase activity were synthesized. Caged substrates were first cleaved by corresponding carboxypeptidases, and then they were activated by luciferase to emit light. Enzymatic activities of biologically important carboxypeptidases can be determined using this technology., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Carboxy (C)-terminal processing proteases (CTP) are a relatively new group of serine proteases. Found in a broad range of organisms - bacteria, archaea, algae, plants and animals - these proteases are involved in the C-terminal processing of proteins. In comparison with amino-terminal processing of bacterial proteins, less is known about C-terminal processing and its physiological function. Bacterial CTPs appear to influence different basal cellular processes. Although CTPs of Gram-negative bacteria are generally referred to as being localized in the periplasm, there is little experimental evidence for this. We show for the first time the subcellular localization of a CTP-3 family protein from Pseudomonas aeruginosa, named CtpA, in the periplasm by a carefully designed fractionation study. Our results provide experimental evidence for the generally accepted hypothesis that CTPs are located in the periplasmic space of Gram-negative bacteria., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Peptidomics is defined as the analysis of peptides present in a tissue extract, usually using mass spectrometry-based approaches. Unlike radioimmunoassay-based detection techniques, peptidomics measures the precise form of each peptide, including post-translational modifications, and can readily distinguish between longer and shorter forms of the same peptide. Also, peptidomics is not limited to known peptides and can detect hundreds of peptides in a single experiment. Quantitative peptidomics enables comparisons between two or more groups of samples and is perfect for studies examining the effect of gene knockouts on tissue levels of peptides. We describe the method for quantitative peptidomics using isotopic labels based on trimethylammonium butyrate, which can be synthesized in five different isotopic forms; this permits multivariate analysis of five different groups of tissue extracts in a single liquid chromatography/mass spectrometry run.

Characterizing the components of GW/processing bodies is key to elucidating RNA interference and messenger RNA processing pathways. This protocol addresses challenges in isolating a low-abundance protein GW182 and GW body (GWB)-associated proteins by building on previous reports that used polyclonal sera containing autoantibodies to GW/P body components. This protocol uses commercially available monoclonal antibodies to GW182 that are covalently coupled to Protein A or G sepharose beads and then used to immunoprecipitate GW182 and associated proteins from cell extracts. Immunoprecipitates are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and probed by western blot with antibodies directed to proteins of interest. This protocol, which is expected to take 4-5 d, provides a biochemical approach for detecting GW182 and associated proteins in biological samples and thus facilitates the elucidation of the diverse functions of GWBs. It is expected that this protocol can be adapted to the detection of other RNA-binding complexes.

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme, consisting of four identical subunits with a M(r) of 43,000. In a previous paper (Occhipinti et al., Biophys J 2003; 85:1165-1175), we developed a structure of the enzyme by molecular modeling and validated it by site-directed mutagenesis and small angle X-ray scattering. Here, we report investigations aimed at further validating the model, as well as at identifying molecular determinants responsible for thermostability. To this end, we took advantage of mass spectrometry techniques, notably LC-MS/MS. The structure was confirmed by such approaches, in that they lead to the identification of a disulfide bridge formed by Cys286 and Cys293, whose location in the model is well suited for giving rise to the crosslink. More notably, we also identified a protease-resistant core consisting of the N- and C-terminal antiparallel alpha-helices, which in the model are predicted to interact with each other via hydrophobic quadrants. On the basis of the model, we also tentatively identified the most tightly interacting residues as Leu7, Ala380, and Leu376. Although the replacement of Ala380 by serine did not detectably impair protein stability, a dramatic drop in thermostability was observed when the two leucines were replaced by either aspartate (L7D; L376D) or asparagine (L7N; L376N). We then investigated the kinetic thermal stability of the wild type and the mutants by determining the thermodynamic activation parameters, DeltaG++, DeltaH++, and DeltaS++. Besides highlighting the key role of the hydrophobic core in thermostability, these results suggest clearly different mechanisms of destabilization by the single mutations, depending on whether the leucines are replaced by asparagines or aspartates., ((c) 2008 Wiley-Liss, Inc.)

Small regulatory RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), or Piwi interacting RNAs (piRNAs) guide regulation of gene expression in many different organisms. The Argonaute (Ago) protein family constitutes the cellular binding partners of such small RNAs and regulates gene expression on the levels of transcription, mRNA stability, or translation. Due to the lack of highly specific and potent monoclonal antibodies directed against the different Ago proteins, biochemical analyses such as Ago complex purification and characterization rely on overexpression of tagged Ago proteins. Here, we report the generation and functional characterization of a highly specific monoclonal anti-Ago2 antibody termed anti-Ago2(11A9). We show that anti-Ago2(11A9) is specific for human Ago2 and detects Ago2 in Western blots as well as in immunoprecipitation experiments. We further demonstrate that Ago2 can be efficiently eluted from our antibody by a competing peptide. Finally, we show that anti-Ago2(11A9) recognizes Ago2 in immunofluorescence experiments, and we find that Ago2 not only localizes to cytoplasmic processing bodies (P-bodies) and the diffuse cytoplasm but also to the nucleus. With the anti-Ago2(11A9) antibody we have generated a potent tool that is useful for many biochemical or cell biological applications.

Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostate-specific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes.

Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through whole live world. Their accurate quantitation in biological samples is important in many fields of biochemistry, not only in routine biochemistry and in fundamental research, but also in clinical and pharmacological research and diagnosis. Since the direct measurement of enzymes by masses is impossible, they must be quantified by their catalytic activities. Many different methods have been applied for this purpose so far. Although photometric methods are undoubtedly the most frequently used, separation methods will further gain their position in this field. The article reviews different possibilities for the assay of enzymatic activity by means of capillary electrophoresis (CE). Both the off-line and on-line enzyme assays based on CE are discussed.

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.

Activins, members of the TGFbeta family of proteins, are widely expressed in a variety of tissues. First identified based on their ability to regulate biosynthesis and secretion of follicle-stimulating hormone (FSH), activins have also been shown to modulate development, cell growth, apoptosis, and inflammation. Despite their many known functions, the precise mechanisms and downstream signaling pathways by which activins mediate their diverse effects remain unknown. We have used a DNA microarray assay to identify genes that are regulated by activin, alone or in combination with gonadotropin-releasing hormone (GnRH), another major regulator of FSH, in a murine gonadotrope-derived cell line (LbetaT2). We used mRNA from these cells to screen Affymetrix Mu74av2 mouse Gene Chip oligonucleotide microarrays, representing approximately 12,400 mouse genes. Treatment of LbetaT2 cells with activin A, a gonadotropin-releasing hormone agonist (GnRHA) or activin A plus GnRHA resulted in alterations in levels of gene expression that ranged in magnitude from 15 to 67-fold. Data analysis identified 268 transcripts that were up- or down-regulated by two-fold or more. Distinct sets of genes were affected by treatment with activin, GnRHA and activin plus GnRHA, suggesting interactions between activin and GnRHA. Changes in expression of seven randomly selected representative genes identified by the microarray technique were confirmed by real-time quantitative PCR and semi-quantitative reverse transcription/PCR (RT/PCR). Modulation of expression of genes by activin suggests that activin may mediate its effects through a variety of signaling pathways.

Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease. CPVL is up-regulated during the maturation of monocytes (MO) to macrophages, although the protein can be seen in both. In primary macrophages, CPVL is glycosylated with high mannose residues and colocalizes with markers for endoplasmic reticulum, while in MO it is more disperse and less clearly associated with endoplasmic reticulum. CPVL is highly expressed in lamellipodia and membrane ruffles, which also concentrate markers of the secretory pathway (MIP-1alpha and tumour necrosis factor-alpha) and major histocompatibility complex (MHC) class I and II molecules. CPVL can be seen on early latex bead and Candida albicans phagosomes, but it is not retained in the maturing phagosome, unlike MHC class I/II. CPVL has a mixed cytosolic and membrane-associated localization but is not detectable on the outer plasma membrane. We propose that CPVL may be involved in antigen processing, the secretory pathway and/or in actin remodelling and lamellipodium formation.

This work was designed to study the expression of the vasodilator peptide angiotensin-(1-7) [Ang-(1-7)] and its generating enzyme (ACE2) in the uteroplacental interface. Placentas were obtained from 11 early pregnancy failures (5 miscarriages and 6 ectopic pregnancies), 15 normotensive, and 10 preeclamptic gestations. In placental villi, the main sites of immunocytochemical expression of Ang-(1-7) and ACE2 were the syncytiotrophoblast, cytotrophoblast, endothelium and vascular smooth muscle of primary and secondary villi. Syncitial Ang-(1-7) expression in samples obtained from miscarriages and ectopic pregnancies was increased compared to normal term pregnancy [2.0 (2.0-2.25 for the 25 and 75% interquartile range) vs 1.3 (1.0-1.9), p<0.01]. In the maternal stroma, Ang-(1-7) and ACE2 were expressed in the invading and intravascular trophoblast and in decidual cells in all 3 groups. Ang-(1-7) and ACE2 staining was also found in arterial and venous endothelium and smooth muscle of the umbilical cord. The expression of Ang-(1-7) and ACE2 was similar in samples obtained from normal term or preeclamptic pregnancies, except for increased expression of ACE2 in umbilical arterial endothelium in preeclampsia [0.5 (0.5-0.8) vs 0.0 (0.0-0.0), p<0.01]. The uteroplacental location of Ang-(1-7) and ACE2 in pregnancy suggests an autocrine function of Ang-(1-7) in the vasoactive regulation that characterizes placentation and established pregnancy.

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The SARS-CoV receptor, human angiotensin 1-converting enzyme 2 (hACE2), was detected in ciliated airway epithelial cells of human airway tissues derived from nasal or tracheobronchial regions, suggesting that SARS-CoV may infect the proximal airways. To assess infectivity in an in vitro model of human ciliated airway epithelia (HAE) derived from nasal and tracheobronchial airway regions, we generated recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF7a/7b) and insertion of the green fluorescent protein (GFP), resulting in SARS-CoV GFP. SARS-CoV GFP replicated to titers similar to those of wild-type viruses in cell lines. SARS-CoV specifically infected HAE via the apical surface and replicated to titers of 10(7) PFU/ml by 48 h postinfection. Polyclonal antisera directed against hACE2 blocked virus infection and replication, suggesting that hACE2 is the primary receptor for SARS-CoV infection of HAE. SARS-CoV structural proteins and virions localized to ciliated epithelial cells. Infection was highly cytolytic, as infected ciliated cells were necrotic and shed over time onto the luminal surface of the epithelium. SARS-CoV GFP also replicated to a lesser extent in ciliated cell cultures derived from hamster or rhesus monkey airways. Efficient SARS-CoV infection of ciliated cells in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.

Biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ACE2) cleaves angiotensin (Ang) II to Ang-(1-7), a heptapeptide identified as an endogenous ligand for the G protein-coupled receptor Mas. No data are currently available that systematically describe ACE2 distribution and activity in rodents. Therefore, we analyzed the ACE2 expression in different tissues of mice and rats on mRNA (RNase protection assay) and protein levels (immunohistochemistry, ACE2 activity, western blot). Although ACE2 mRNA in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ACE2, as also demonstrated in the kidney and colon. Corresponding to mRNA, ACE2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. Contrary to mRNA, we found weak activity in the lung of both species. Our data demonstrate a tissue- and species-specific pattern for ACE2 under physiological conditions.

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.

Angiotensin-converting enzyme 2 (ACE2) is a recently discovered homologue of angiotensin-converting enzyme (ACE) that is thought to counterbalance ACE. ACE2 cleaves angiotensin I and angiotensin II into the inactive angiotensin 1-9, and the vasodilator and anti-proliferative angiotensin 1-7, respectively. ACE2 is known to be present in human kidney, but no data on renal disease are available to date. Renal biopsies from 58 patients with diverse primary and secondary renal diseases were studied (hypertensive nephropathy n = 5, IgA glomerulopathy n = 8, minimal change nephropathy n = 7, diabetic nephropathy n = 8, focal glomerulosclerosis n = 5, vasculitis n = 7, and membranous glomerulopathy n = 18) in addition to 17 renal transplants and 18 samples from normal renal tissue. Immunohistochemical staining for ACE2 was scored semi-quantitatively. In control kidneys, ACE2 was present in tubular and glomerular epithelium and in vascular smooth muscle cells and the endothelium of interlobular arteries. In all primary and secondary renal diseases, and renal transplants, neo-expression of ACE2 was found in glomerular and peritubular capillary endothelium. There were no differences between the various renal disorders, or between acute and chronic rejection and control transplants. ACE inhibitor treatment did not alter ACE2 expression. In primary and secondary renal disease, and in transplanted kidneys, neo-expression of ACE2 occurs in glomerular and peritubular capillary endothelium. Further studies should elucidate the possible protective mechanisms involved in the de novo expression of ACE2 in renal disease., (Copyright (c) 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P(2) and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P(2) via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P(2)- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.

Previously we demonstrated that kidney concentration and urinary excretion of angiotensin-(1-7) are increased during normal pregnancy in rats. Since this finding may reflect local kidney production of angiotensin-(1-7), we determined the immunocytochemical distribution of angiotensin-(1-7) and its newly described processing enzyme, ACE2, in kidneys of virgin and 19-day-pregnant Sprague-Dawley rats. Sprague-Dawley rats were killed at the 19th day of pregnancy, and tissues were prepared for immunocytochemical by using a polyclonal antibody to angiotensin- (1-7) or a monoclonal antibody to ACE2. Angiotensin-(1-7) immunostaining was predominantly localized to the renal tubules traversing both the inner cortex and outer medulla. ACE2 immunostaining was localized throughout the cortex and outer medulla and was visualized in the renal tubules of both virgin and pregnant rats. The quantification of angiotensin-(1-7) and ACE2 immunocytochemical staining showed that in pregnant animals, the intensity of the staining increased by 56% and 117%, respectively (P<0.05). This first demonstration of the immunocytochemical distribution of angiotensin-(1-7) and ACE2 in kidneys of pregnant rats shows that pregnancy increases angiotensin-(1-7) immunocytochemical expression in association with increased ACE2 intensity of staining. The findings suggest that ACE2 may contribute to the local production and overexpression of angiotensin-(1-7) in the kidney during pregnancy.

Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins and exert important biological functions. Assays for carboxypeptidase activity that rely on change of absorbance generally suffer from low sensitivity and are difficult to adapt to high-throughput screening. We have developed a sensitive, robust assay for basic carboxypeptidase activity that makes use of electrochemiluminescent (ECL) detection of reaction product. In this assay, a peptide substrate contains the epitope for antibody (G2-10) binding which is masked by a C-terminal arginine. Carboxypeptidase activity exposes the epitope, allowing the binding of ruthenylated G2-10 which is then detected using ECL. High sensitivity allowed detection limits of 1-2 pM enzyme for carboxypeptidase B and activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The inhibition of several basic carboxypeptidases by commercially available inhibitors was studied. This antibody-based method can be extended to other sensitive detection techniques such as amplified luminescent proximity homogeneous assay. The high sensitivity of the assay allowed the determination of the activatable levels of TAFI in human and other animal plasma in the presence of epsilon -aminocaproic acid, an active-site inhibitor that stabilizes TAFIa. A method to isolate in situ activated TAFIa from human serum in the presence of epsilon -aminocaproic acid was also developed.

ACE2, initially cloned from a human heart, is a recently described homologue of angiotensin-converting enzyme (ACE) but contains only a single enzymatic site that catalyzes the cleavage of angiotensin I to angiotensin 1-9 [Ang(1-9)] and is not inhibited by classic ACE inhibitors. It also converts angiotensin II to Ang(1-7). Although the role of ACE2 in the regulation of the renin-angiotensin system is not known, the renin-angiotensin system has been implicated in the pathogenesis of diabetic complications and in particular in diabetic nephropathy. Therefore, the aim of this study was to assess the possible involvement of this new enzyme in the kidney from diabetic Sprague-Dawley rats to compare and contrast it to ACE. ACE2 and ACE gene and protein expression were measured in the kidney after 24 weeks of streptozocin diabetes. ACE2 and ACE mRNA levels were decreased in diabetic renal tubules by approximately 50% and were not influenced by ACE inhibitor treatment with ramipril. By immunostaining, both ACE2 and ACE protein were localized predominantly to renal tubules. In the diabetic kidney, there was reduced ACE2 protein expression that was prevented by ACE inhibitor therapy. The identification of ACE2 in the kidney, its modulation in diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of various angiotensin peptides provides a rationale to further explore the role of this enzyme in various pathophysiological states including diabetic complications.

We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic beta-cell line derived from the Cpe(fat)/Cpe(fat) mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.

RT-nested PCR has been introduced as a highly specific and sensitive assay method to detect the prostate-specific membrane antigen (PSM) mRNA in peripheral blood. However, appreciable percentages of false-positive cases have been reported. Additionally, primer sets reported previously could not discriminate between PSM and PSM', an alternatively spliced variant, mRNA. These isoforms can be produced from a single gene. Switches in alternative splicing patterns are often controlled with strict cell-type or developmental-stage specificity. Therefore, it is most important to discriminate between PSM mRNA and PSM' mRNA. Using our highly specific primer sets, PSM mRNA was detected in 3 of 24 peripheral blood samples of normal male volunteers (12.5%) and was not detected in peripheral blood of 11 normal female volunteers. PSM' mRNA was detected in 5 of 24 peripheral blood samples of normal male volunteers (20.8%) and in 4 of 11 of normal female volunteers (36.4%). PSM' mRNA induced false-positive results, it is important for genetic diagnosis of prostate cancer to discriminate between PSM and PSM' using our primer sets with high specificity. The advances in the uniquely designed primer sets may allow researchers to detect a real PSM mRNA without PSM' mRNA.

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.

A time-resolved fluorescence resonance energy transfer (TR-FRET) detection method based on the formation of a PDZ domain.peptide ligand complex has been developed for enzymatic assays as an alternative to immuno-based detection strategies. The enzyme substrate is a "masked" biotinylated PDZ domain peptide ligand containing the consensus sequence Ser-X-Val-COOH. The critical residues in the binding consensus sequence of the ligand have been modified, for example, by phosphorylation of Ser or C-terminal extensions, providing binding-incompetent PDZ domain peptides. On processing by the corresponding enzyme, the binding epitope is exposed, and the product sequence is recognized specifically by Eu(3+) chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ) (GST-PDZ-glutathione S-transferase fused to PDZ domain). A ternary complex is subsequently formed by addition of allophycocyanin-labeled streptavidin ([XL665]SA), which binds to the biotinylated N terminus of the peptide, and detected by TR-FRET. Reported here are examples of the applicability of this detection strategy to three enzymatic systems, an endoprotease, an exoprotease, and a Ser/Thr phosphatase., (©2002 Elsevier Science (USA).)

Prostate cancer remains the most common cancer type in men in the United States. Efforts are increasing to evaluate and to discover diagnostic and therapeutic markers for prostate cancer patients. One of these, prostate-specific membrane antigen (PSMA), is a transmembrane protein highly expressed in all types of prostatic tissue, especially cancer. The radio-immunoconjugate form of the anti-PSMA monoclonal antibody (mAb) 7E11, known as the ProstaScint scan, is currently being used to diagnose prostate cancer metastasis and recurrence. Early promising results from various Phase I and II trials have utilized PSMA as a therapeutic target. Recently, PSMA expression in endothelial cells of tumor-associated neovasculature has been described. PSMA's possible role in malignant angiogenesis newly expands the realm of its possible beneficial uses, especially as new anti-PSMA mAbs continue to be developed and refined.

Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemia-induced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brain's response to ischemia.

Entry of duck hepatitis B virus (DHBV) is initiated by specific interaction of its large envelope protein (L) with a cellular entry receptor, recently identified as carboxypeptidase D (CPD; historically gp180). In this report, we present evidence demonstrating that this receptor is down-regulated as a result of DHBV infection: (i) receptor levels determined by Western blot were much reduced in DHBV-infected duck livers and undetectable by immunostaining in infected cultured hepatocytes; (ii) results from metabolic labeling experiments indicate enhanced receptor protein turnover; (iii) the kinetics of receptor loss from newly infected cells correlated with the accumulation of newly synthesized viral protein; (iv) expression of DHBV L protein, transduced from a recombinant adenovirus, was sufficient to eliminate gp180/CPD from the Golgi compartment, its normal predominant location; (v) gp180/CPD remained absent from the Golgi compartment in infected hepatocytes, even after overexpression from a recombinant adenovirus, while residual amounts subsequently became detectable in a perinuclear compartment, containing DHBV L protein; (vi) expression of DHBV L protein in a HepG2 cell line, stably expressing gp180/CPD, leads to incomplete receptor maturation and induces its degradation. Taken together, these data are consistent with a model in which the virus receptor interacts early in the biosynthetic pathway with the viral L protein, leading to its retention in a pre-Golgi compartment and to subsequent degradation, thus preventing receptor interference with the export of DHBV via the secretory pathway which it shares with its receptor. Accordingly, and analogously with receptor down-regulation in retroviral systems, DHBV receptor down-modulation may account for the much-reduced efficiency of DHBV superinfection of preinfected hepatocytes.

Prostate-specific membrane antigen (PSMA) is a potential target in prostate cancer patients because it is very highly expressed and because it has been reported to be upregulated by androgen deprivation. This review discusses the historical background, biochemical characteristics, gene regulation, potential for targeting, tissue localization, and a novel T-body strategy.

Background: Patients with seminal vesicle invasion (SVI) at radical retropubic prostatectomy (RRP) have a poor prognosis. Routine microscopic examination of pelvic lymph nodes (LNs) can miss small metastases and, thereby, confuse tumor staging and clinical decision-making. The authors used immunohistochemical and molecular methods to examine archival paraffin-embedded LNs of men who had undergone RRP for clinically localized prostate carcinoma and who had tumors demonstrating SVI and negative LNs at surgery., Methods: Between June 1982 and June 1997, 2151 consecutive men underwent RRP for clinically localized prostate carcinoma. Of these, 109 (5.1%) tumors had SVI with negative LNs. The actuarial likelihood of having a tumor that was undetectable by testing prostate-specific antigen (PSA) 5 and 10 years after surgery was 45% and 29%, respectively, for men with isolated SVI. Archival LN specimens were available for 102 men who had isolated SVI. Reverse transcription polymerase chain reaction (RT-PCR) was performed for PSA and prostate-specific membrane antigen (PSMA). All specimens were examined concurrently by immunohistochemistry (IHC)., Results: Careful reevaluation of pelvic LNs demonstrated metastases in 9 (8.8%) men originally classified as metastasis-free. Reevaluation by hematoxylin and eosin (H&E) staining identified three previously unrecognized cases of LN metastases. IHC identified six cases, three of which were missed by H&E. RT-PCR identified four cases, three of which were not revealed by other methods., Conclusions: The poor prognosis of patients with SVI does not seem due to occult LN metastases. The low yield of unsuspected foci of prostate carcinoma in the LNs of men with SVI and negative LNs by routine staging does not justify IHC or molecular examination to find occult carcinoma., (Copyright 2000 American Cancer Society.)

To study early events in mast cell / basophil development, the phenotype of a panel of murine cell lines at various stages of differentiation was determined. Based on the expression on various mast cell-specific proteases and several additional hematopoietic differentiation markers, the cell lines CFTL-15 and MCP5 / L were clearly identified as mast cells, although with a relatively immature phenotype. These two cell lines express the high-affinity IgE receptor alpha-chain, the mouse mast cell protease (MMCP)-5 and the carboxypeptidase A (CPA). Bone marrow-derived mast cells and the transplantable mast cell tumor MTC were shown to express the IgE receptor alpha-chain, MMCP-5 and CPA, as well as the mast cell tryptase MMCP-6 and the chymase MMCP-4, a protease expressed only during late stages of mast cell differentiation. These two cell types thus display a more mature mast cell phenotype. In contrast, the cell lines P815 and 32D cl3 did not express any mast cell differentiation markers. Interestingly, the IC-2 cell line was shown to express several markers for immature mast cells and in addition MMCP-8, a serine protease which may represent a marker for mouse basophils. By antibody staining, almost all IC-2 cells were shown to express MMCP-8. This indicates that individual cells may simultaneously express both mast cell and basophil markers. Moreover, these findings suggest that an early branch point in hematopoietic development where mast cells and basophils have a common precursor cell may exist.

Background: We tested the ability of the nested reverse transcription polymerase chain reaction (RT-PCR) assay to detect signs of biochemical recurrence of prostate cancer in the lymph nodes and peripheral blood of patients with pT3N0 prostate cancer., Methods: Using lymph nodes and pre- and postoperative peripheral blood dissected from 30 patients with pT3N0 prostate cancer treated by radical prostatectomy, we used RT-PCR for prostate-specific membrane antigen (PSM) and serum prostate-specific antigen (PSA) to determine the presence of prostate cancer. Results of the nested RT-PCR assay were compared with pathological stages and biochemical recurrence., Results: Two of 13 patients with capsular penetration (15%), 6 of 10 patients with invasion of seminal vesicles (60%), and 3 of 7 patients with a positive surgical margin (43%) were RT-PCR-positive for PSM and/or PSA in the lymph nodes. Results of preoperative RT-PCRs of peripheral blood for PSM and for PSA significantly differed between positive and negative results of RT-PCR in lymph nodes (P < 0.001 and P < 0.001, respectively). Results of postoperative RT-PCRs of peripheral blood for PSM and for PSA also significantly different between positive and negative results of RT-PCR in lymph nodes (P = 0.011 and P = 0.001, respectively). Nine of 11 patients with positive nested RT-PCR for PSM and/or PSA in the lymph nodes (82%) experienced biochemical recurrence. Significant difference in Kaplan-Meier recurrence-free actuarial curves was noted between patients who nested positive and negative on RT-PCR in the lymph nodes, pre- and postoperative peripheral blood, biopsy and prostatectomy Gleason score, and preoperative PSA values. In multivariate analysis, biopsy and prostatectomy Gleason score (P = 0.026, P = 0.020, respectively), pre- and postoperative RT-PCR for PSM in peripheral blood (P = 0.030 and P = 0.040, respectively), and RT-PCR for PSM in lymph nodes (P = 0.035) were independent prognostic factors., Conclusions: Nested RT-PCR assay of the lymph nodes or peripheral blood significantly predicted biochemical recurrence after surgery. It may help identify patients at risk for recurrence and progression of prostate cancer., (Copyright 2000 Wiley-Liss, Inc.)

Studies on the developing mammalian pancreas have suggested that insulin and glucagon co-exist in a transient cell population and that peptide YY (PYY) marks the earliest developing endocrine cells. We have investigated this in the embryonic avian pancreas, which is characterised by anatomical separation of insulin and glucagon islets. Moreover, we have compared the development of the endocrine cells to that of processing enzymes involved in pancreatic hormone biosynthesis. PYY-like immunoreactivity occurred in islet cells from the youngest stages examined: it increased in amount from approximately 5 days of incubation and was co-localised with glucagon and to a lesser extent with insulin. Insulin and glucagon cells were numerous: co-existence of the two peptides in the same cells was but rarely observed. From the youngest stages examined, prohormone convertase (PC) 1/3-like immunoreactivity was detected in insulin cells and PC2-, 7B2- and carboxypeptidase E-like immunoreactivity in both glucagon and insulin cells. We conclude that: (1) PYY-like immunoreactivity occurs in avian islet cells but generally in lesser amounts than in mammals at the earlier stages, (2) the paucity of cells co-expressing insulin and glucagon indicate that all avian insulin cells do not pass through a stage where they co-express glucagon and (3) the early expression of the enzymes responsible for the processing of prohormones suggests that this process is initiated soon after islet cells first differentiate.

The effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 microg ethinylestradiol, 150 microg levonorgestrel) or third generation OC (30 microg ethinylestradiol, 150 microg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-alpha2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor- (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.

We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification). Our method is simpler, less time-consuming, and suitable for multiple, simultaneous determinations and the substrate bound to nitrocellulose can be stored for several months without significant alteration of its properties. Peptidases other than tubulin carboxypeptidase can act on [14C]tyrosinated tubulin bound to nitrocellulose, solubilizing radioactive compounds, suggesting the eventual applicability of this method to assay proteases in general. Other features and advantages of the assay as well as its limitations are discussed., (Copyright 2000 Academic Press.)

The evaluation of new therapies in prostate cancer requires unique end points for agents with diverse mechanisms of action. Because retinoic acid may have a confounding effect on prostate-specific antigen, we incorporated a pathological end point into the outcome assessment of two sequential clinical trials using all-trans-retinoic acid (ATRA) and the combination of 13-cis-retinoic acid and IFN-2a (cRA¿IFN). Pre- and posttherapy tumor biopsy specimens were studied for histological changes, apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay), and proliferation index (Ki67). Prostate-specific membrane antigen (PSMA) expression was also evaluated using two different monoclonal antibodies to its intracellular domain (Cytogen 7E11 and Hybritech PM2). Fourteen patients with androgen-independent disease were treated with ATRA (50 mg/m2 p.o. every 8 h daily) and 16 androgen-independent and 4 androgen-dependent patients were treated with cRA¿IFN (10 mg/kg/day cRA plus 3, 6, or 9 million units daily IFN). Both therapies were well tolerated, with fatigue and cheilitis being the most common adverse events. Clinical activity, assessed by radiographs and serum prostate-specific antigen, was minimal, and the majority of patients progressed within 3 months. One patient with androgen-dependent disease had prolonged stabilization for >1 year. The majority of cases (95%) showed no gross histological changes and no difference in apoptotic or proliferative indices. Increased PSMA immunoreactivity was seen in seven of nine (78%) cases using PM2 antibody and in two of nine (22%) cases using the 7E11 antibody. Although antitumor effects were modest, the results suggest a role for retinoids in modulating the expression of PSMA on prostate cancer cells.

Background: Prostate specific membrane antigen (PSMA) is a transmembrane glycoprotein highly expressed in benign prostate secretory-acinar epithelium and prostate carcinoma. The results of several studies suggest that PSMA expression is increased in prostate carcinoma cell lines subjected to androgen deprivation and in androgen-independent tumors. The authors studied the effects of short term (3-month) androgen deprivation on PSMA expression in prostate carcinoma specimens using two anti-PSMA monoclonal antibodies (mAbs), 7E11 and PM2J004.5., Methods: The study included patients with clinically localized prostate carcinoma who were prospectively randomized into 1 of 2 treatment groups: 3 months of neoadjuvant androgen deprivation therapy followed by radical prostatectomy (ADT/RP), or radical prostatectomy (RP) alone. Representative formalin fixed, paraffin embedded prostate sections were immunostained with the anti-PSMA mAbs 7E11 and PM2J004.5 by the streptavidin-biotin method. The authors recorded the staining intensity and the percentage of positive cells stained in benign epithelium, high grade prostatic intraepithelial neoplasia (PIN), and prostate carcinoma. They compared the results of 7E11 with those of PM2J004.5 in benign epithelium, high grade prostate, and carcinoma and also compared the results between the two treatment groups (ADT/RP vs. RP alone)., Results: Both anti-PSMA mAbs stained benign secretory-acinar epithelium, high grade PIN, and prostate carcinoma. In both treatment groups, PM2J004.5 reacted with a significantly greater percentage of cells (P < 0.001) and with significantly greater intensity (P < 0.001) compared with 7E11 in benign epithelium and prostate carcinoma. With both anti-PSMA mAbs, the percentage of cells stained and the intensity of staining in high grade PIN was similar to that in prostate carcinoma. In the group that received RP alone, the percentage of cells stained and the intensity of staining with 7E11 were significantly greater in high grade PIN and prostate carcinoma compared with benign epithelium (P < 0.001), and the intensity of staining with the PM2J004.5 was significantly greater in high grade PIN and prostate carcinoma compared with benign epithelium (P < 0.001). In the ADT/RP group, the percentage of cells stained and the intensity of staining with 7E11 and PM2J004.5 were significantly greater in prostate carcinoma compared with benign epithelium (P < 0.006). PSMA staining did not correlate with either Gleason score (in the group that received RP alone) or pathologic stage (in both the RP-alone and ADT/RP groups) and did not differ between the two treatment groups., Conclusions: Short term neoadjuvant ADT does not affect PSMA expression in benign prostate secretory-acinar epithelium, high grade PIN, or prostate carcinoma. Prostate carcinoma and high grade PIN express significantly higher levels of PSMA than benign prostate secretory-acinar epithelium. Compared with 7E11, the PM2J004.5 anti-PSMA mAb is a more sensitive immunohistochemical marker of prostate carcinoma in formalin fixed, paraffin embedded tissue., (Copyright 2000 American Cancer Society.)

The receptor molecules for human and animal hepatitis B viruses have not been defined. Previous studies have described a 170 to 180 kDa molecule (p170 or gp180) that binds in vitro to the pre-S domain of the large envelope protein of duck hepatitis B virus (DHBV); cDNA cloning revealed the binding protein to be duck carboxypeptidase D (DCPD). In the present study, the DCPD cDNA was transfected into several nonpermissive human-, monkey-, and avian species-derived cell lines. Cells transfected with a plasmid encoding the full-length DCPD protein bound DHBV particles, whereas cells expressing truncated versions of DCPD protein that fail to bind the pre-S protein did not. The DHBV binding to DCPD-reconstituted cells was blocked by a monoclonal antibody that neutralizes DHBV infection of primary duck hepatocytes (PDH) and also by a pre-S peptide previously shown to inhibit DHBV infection of PDH. In addition to promoting virus binding, DCPD expression was associated with internalization of viral particles. The entry process was prevented by incubation of reconstituted cells with DHBV at 4 degrees C and by the addition of energy-depleting agents known to block DHBV entry into PDH. These results demonstrated that DCPD is a DHBV receptor. However, the lack of complete viral replication in DCPD-reconstituted cells suggested that additional factors are required for postentry events in immortalized cell lines.