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E Wilk

Publications and source records attributed to E Wilk.

18 recordsLinked to original sources
PubMed1999

Altered donor and recipient Ly49+ NK cell subsets in allogeneic H-2d --> H-2b and H-2b --> H-2d bone marrow chimeras.

NK cells reject non-self hematopoietic bone marrow (BM) grafts via Ly49 receptor-mediated MHC class I-specific recognition and calibration of receptor expression levels. In this paper we investigated how Ly49+ subset frequencies were regulated dependent on MHC class I expression. The development of donor and host Ly49A+ (recognizes H-2Dd and H-2Dk ligands) and Ly49C/I+ (Ly49CBALB/c recognizes H-2Kb, H-2Kd, and H-2Dd, and Ly49CB6 recognizes only H-2Kb) NK cell frequencies were monitored for 120 days in murine-mixed allogeneic BM chimeras. C57BL/6 (H-2b) BM was transplanted into BALB/c (H-2d) mice and vice versa. Peripheral NK cell populations were examined every 5 days. Chimerism was found to be stable with 80-90% donor NK cells. In contrast to syngeneic controls reexpressing pretransplant patterns, donor and host NK cells revealed new and mainly reduced subset frequencies 55 days after allogeneic transplantation. Recipient NK cells acquired these later than donor NK cells. In H-2d --> H-2b chimeras Ly49A+, Ly49C/I+, and Ly49A+/Ly49C/I+ proportions were mainly diminished upon interaction with cognate ligands. Also in H-2b --> H-2d chimeras, Ly49A+ and Ly49A+/Ly49C/I+ subsets were reduced, but there was a transient normalization of Ly49C/I+ proportions in the noncognate host. After 120 days all subsets were reduced. Therefore, down-regulation of developing Ly49A+ and Ly49C/I+ chimeric NK cell frequencies by cognate ligands within 7-8 wk after BM transplantation may be important for successful engraftment.

Animals↗
PubMed1998

Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase.

An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4. 11.7) was purified to near homogeneity from rabbit brain cytosol. Its properties were similar to an enzyme described previously (Kelly, J. A., Neidle, E. L., and Neidle, A. (1983) J. Neurochem. 40, 1727-1734). Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates. Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5). The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis. The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags. Human and mouse clones described as "similar to a yeast vacuolar aminopeptidase" and containing full-length cDNAs were identified and sequenced. The human cDNA was expressed in Escherichia coli. The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases. Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates. Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism.

Amino Acid Sequence↗
PubMed1998

Production and properties of a recombinant soluble form of aminopeptidase A.

Aminopeptidase A is a homodimeric membrane-bound zinc metallopeptidase anchored at the plasma membrane by a 22-amino-acid hydrophobic segment. The anchor segment separates a small N-terminal cytoplasmic domain from a large ectodomain that contains the active site. Site-directed mutagenesis was performed to investigate the role of the cytoplasmic domain of aminopeptidase A in membrane anchoring and routing of the enzyme. Expression in COS-7 cells of a mutant lacking the N-terminal cytoplasmic domain resulted in the efficient secretion of a catalytically active enzyme in the medium. The soluble mutated aminopeptidase A, purified from the medium of a stable cell line, exhibited similar biochemical features to those of the wild-type enzyme. Pulse/chase metabolic labeling experiments revealed that the soluble form is generated intracellularly at an early stage of biosynthesis, suggesting that the signal peptide/membrane anchor domain of aminopeptidase A is removed in the endoplasmic reticulum through the action of the signal peptidase.

Amino Acid Sequence↗
PubMed1996

Inhibition of prolyl oligopeptidase by Fmoc-aminoacylpyrrolidine-2-nitriles.

Prolyl oligopeptidase (EC 3.4.21.26), a widely distributed cytosolic enzyme, cleaves peptidylprolyl peptide and peptidylprolyl amino acid bonds in many neuropeptide substrates. Its action on vasopressin has been proposed as the underlying mechanism accounting for the ability of inhibitors of prolyl oligopeptidase to reverse scopolamine-induced amnesia in rats. Future behavioral studies would be facilitated by the availability of potent inhibitors readily synthesized from common intermediates. A series of Fmoc-aminoacylpyrrolidine-2-nitriles prepared by a simple two-step synthesis were found to be potent noncompetitive inhibitors of the rabbit brain enzyme. The most potent inhibitors, Fmoc-prolyl-pyrrolidine-2-nitrile and Fmoc-alanyl-pyrrolidine-2-nitrile, each have a Ki of 5 nM. The compounds are cell permeable and stable. They do not inhibit the related enzyme dipeptidyl peptidase IV (EC 3.4.14.5). When administered intraperitoneally to mice, Fmoc-alanyl-pyrrolidine-2-nitrile crosses the blood-brain barrier to inhibit brain prolyl oligopeptidase. The ease of synthesis, potency, efficacy in vivo, stability, and specificity of Fmoc-aminoacylpyrrolidine-2-nitriles may make them inhibitors of choice in studies probing the physiological significance of prolyl oligopeptidase.

Animals↗
PubMed1996

[Selected chemical compounds as potential sources of massive poisoning. Physicochemical properties, poisoning symptoms, treatment].

Characteristics of 42 chemical compounds (17 gases and 24 liquids) sustaining potential danger of massive poisonings in case of accident or chemical disaster is presented. Description of each substance includes: physiocochemical properties, pathophysiology and symptoms of acute poisonings with those chemicals. Rules and procedures of treatment are presented including premedical first aid and medical treatment.

Accidents↗
PubMed1995

Aminoacylpyrrolidine-2-nitriles: potent and stable inhibitors of dipeptidyl-peptidase IV (CD 26).

Dipeptidyl-peptidase IV (EC 3.4.14.5) also known as CD26 is a membrane-bound serine peptidase which cleaves N-terminal dipeptides from a peptide chain containing a proline residue in the penultimate position. The enzyme is believed to play an important role in neuropeptide metabolism and T-cell activation. A series of aminoacylpyrrolidine-2-nitriles, in which the carboxyl group of proline is replaced by a nitrile group, was synthesized as inhibitors of dipeptidyl-peptidase IV. All compounds were found to competitively inhibit a homogeneous preparation of the rat kidney enzyme with Ki values in the low to submicromolar range. The nitriles presumably react with the active-site serine to form an imidate adduct. The compounds were stable following incubation either for 20 h at 37 degrees C or 72 h at room temperature. They proved to be poor inhibitors of dipeptidyl-peptidase II and prolyl oligopeptidase. These studies demonstrate that the generally held concept that nitriles are poor inhibitors of serine proteinases needs to be reconsidered. Amino-acylpyrrolidine-2-nitriles by virtue of their ease of synthesis, stability, specificity, and inhibitory potency appear to be superior to other described dipeptidyl-peptidase IV inhibitors.

Animals↗
PubMed1994

Organization, sequence, and function of 34.5 kb of genomic DNA encompassing several murine T-cell receptor alpha/delta variable gene segments.

A cosmid-bearing murine genomic DNA, encompassing several T-cell receptor variable alpha/delta gene segments, has been sequenced using shotgun DNA cloning methodology coupled with fluorescence-based high-through-put DNA sequencing technology. This region, spanning 34.5 kb, contains a pseudogene V alpha gene segment (psi V alpha 16.1), a V delta gene segment (V delta 2), and a variable gene segment that has been reported to be expressed with both C alpha (V alpha 6) and C delta (V delta 3). Therefore, this cosmid is the ideal vehicle for examining the possible control sequences that may be involved in determining whether a V gene segment associates with either C alpha or C delta. Polymerase chain reaction experiments demonstrate that the two functional variable gene segments (V delta and V alpha/delta) are expressed individually with both C alpha and C delta genes as mRNA, indicating a permissiveness in their expression patterns. In addition, a variety of genomic sequence-related features have been identified.

Amino Acid Sequence↗
PubMed1994

Rat kidney glutamyl aminopeptidase (aminopeptidase A): molecular identity and cellular localization.

Glutamyl aminopeptidase [aminopeptidase A (EAP), EC 3.4.11.7] is an ectoenzyme that selectively hydrolyzes acidic amino acid residues from the amino terminus of oligopeptides. EAP activity is highest within the kidney and small intestine. The murine pre-B cell BP-1/6C3 and the human kidney glycoprotein gp160 differentiation antigens have been reported to have biochemical properties indistinguishable from EAP. It is not known, however, if rat kidney EAP is a homologue of these antigens or molecularly distinct. Using the reverse transcription-polymerase chain reaction method with oligonucleotide primers based on the BP-1/6C3 nucleotide sequence, we isolated a 450-bp partial cDNA from rat kidney poly(A)+ RNA. The partial cDNA encoded a predicted protein that was 92% and 86% identical to the murine BP-1/6C3 and human gp160 antigens, respectively; the amino acid sequence within the zinc-binding domain was completely conserved. Purification of EAP from rat kidney and microsequence analysis of a tryptic digest peptide fragment (18-mer) indicated that the fragment was highly similar to a region within the BP-1/6C3 and gp160 proteins. Northern blot hybridization and immunoblot analyses were also consistent with labeling of products the same size as reported for the BP-1/6C3 and gp160 antigens. There was a good correlation between the cellular distribution of EAP mRNA and EAP immunoreactivity, with proximal tubules and glomerular mesangial cells having the highest densities. These results indicate that rat kidney EAP is a species homologue of the murine BP-1/6C3 and human gp160 antigens. Furthermore, on the basis of its cellular localization, rat kidney EAP is likely to be involved in degradation of oligopeptides within the glomerulus and the glomerular filtrate. Since cells that express EAP also express receptors for angiotensin II, an intrarenal vasoactive hormone that is a substrate for EAP, these results further suggest that EAP may play a role in modulating the activity of intrarenal angiotensin II.

Amino Acid Sequence↗
PubMed1993

Localization of immunoreactive glutamyl aminopeptidase in rat brain. I. Association with cerebral microvessels.

Glutamyl aminopeptidase (aminopeptidase-A, EC 3.4.11.7) is an ectoenzyme that selectively hydrolyses N-terminal glutamyl and aspartyl residues from oligopeptides, including (Asp1) angiotensin II. Here we sought to determine the distribution of glutamyl aminopeptidase (EAP) in rat brain. EAP was purified to homogeneity from rat kidney and polyclonal antiserum raised in rabbits. Immune serum inhibited EAP enzyme activity in kidney homogenates and labeled two major protein bands of M(r) = 136,000 and M(r) = 101,000 in immunoblots of kidney protein. EAP-like immunoreactivity was concentrated on kidney proximal tubule brush borders. Immunocytochemical staining of rat brain indicated that EAP-like immunoreactivity was primarily associated with cerebral microvessels. Positive staining was detected in microvessels ranging in size from capillaries up to vessels approximately 50 microns in diameter. Isolated cerebral microvessels had a 23-fold enrichment in EAP enzyme activity (193.1 +/- 40.4 nmol/mg protein/h) compared to brain homogenates. Finally, immunoblots of isolated cerebral microvessels resulted in a pattern of labeling similar to that seen with kidney homogenates. These results indicate that EAP activity in brain is primarily associated with cerebral microvessels, and suggest that EAP may be involved in the metabolism of circulating or locally formed peptides.

Aminopeptidases↗
PubMed1984

A sensitive procedure for determination of cathepsin D: activity in alveolar and peritoneal macrophages.

Several new synthetic substrates fulfilling the specificity requirements of cathepsin D were synthesized. One of these D-Phe-Ser(O-CH2-C6H5)-Phe-Phe-Ala-Ala-pAB(pAB = p-aminobenzoate) proved to be highly sensitive and convenient for measuring activity. Enzyme determination was carried out in a two-step reaction. In the first step the enzyme hydrolyzes the Phe-Phe bond of the substrate at pH 3.4. In the second step aminopeptidase M (EC 3.4.11.2) degrades one of the products Phe-Ala-Ala-pAB at pH 7 to 8 with the release of free pAB, which is then determined by a diazotization procedure. Activity can be measured in as little as 1 to 5 micrograms of macrophage protein. The activity of cathepsin D in rat alveolar macrophages was almost ten times higher than in resident peritoneal macrophages, and more than 25 times higher than in blood monocytes. The data indicate that transformation of blood monocytes into macrophages is associated with a much greater increase of cathepsin D activity in alveolar than peritoneal macrophages.

Aminopeptidases↗
PubMedDate not supplied

[Inhalation heat shock].

Using the example of a recent case, we examine the possibilities of trace analysis and fire protection techniques in the investigation of fire deaths. The effective cooperation between forensic pathologists and fire inspectors is presented and we discuss the mathematical-physical uses of fire simulation calculations. We review the trace analysis results and discuss the variety of technical possibilities of modern engineering techniques for fire protection.

Adolescent↗