Evidence for an essential histidine in neutral endopeptidase 24.11

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.     

The need for immune evaluation prior to thymosin-containing chemoimmunotherapy for melanoma

Summary In an attempt improve the response to BCG or BCG + DTIC therapy in Stage IIIB melanoma patients, we added thymosin treatment with repeated doses of 4 mg/m² or 40 mg/m². Twenty-eight patients were clinically and immunologically evaluable. Pretreatment immunological evaluation consisted of determination of delayed-type hypersensitivity to recall antigens, enumeration of E-rosettes in blood, and measurement of blood lymphocyte response to phytohemmagglutinin (PHA) and concanavalin A (Con-A). The disease-free interval was correlated with thymosin dose and parameters of immunocompetence. Immunocompetent melanoma patients treated with a high thymosin dose and BCG relapsed earlier than those treated with a low thymosin dose and BCG. Thus, 72% of the melanoma patients who had a PHA SI50 and were treated with 4 mg thymosin/m², were disease-free at 9 months, compared with only 31% of those treated with 40 mg/m² (P=0.02). When the dermatophytin delayed hypersensitivity response (>10 mm induration) was used as a parameter of immunocompetence, 86% of the patients treated with 4 mg/m², were disease-free at 9 months, as against 28% of patients treated with 40 mg thymosin/m² (P=0.07). None of the immunoincompetent patients on high thymosin dose relapsed (0/3). The results suggest that while a high thymosin dose (40 mg/m²) may be detrimental to immunocompetent patients, it may have a beneficial effect in immunoincompetent patients. A low thymosin dose is probably not detrimental to immunocompetent melanoma patients in this study. Monitoring of the immune status prior to and during the use of thymosin in cancer immunotherapy is mandatory.This paper was presented at the Thirteenth Annual Meeting of the American Society for Clinical Oncology, Denver, 1977     

Pseudomonas vaccine: A phase I evaluation for cancer research

Summary A heptavalent lipopolysaccharide vaccine of Pseudomonas aeruginosa (Pseudogen) was administered at four dose levels (0.1, 0.25, 0.5, and 0.75 mg/m²) to patients with advanced metastatic cancer that had proved refractory to chemotherapy. The vaccine was administered subcutaneously twice weekly. Local toxicity was seen in erythema, edema, pain and tenderness at the site of injection, and painful regional lymphadenopathy; manifestations of systemic toxicity included fever, chills, myalgias, nausea, and vomiting. Toxicity showed a clear-cut dose dependence. The maximally tolerated dose by this route and schedule was 0.5 mg/m². A significant rise of antibody titers was observed at all four dose levels. Evaluation of the delayed cutaneous hypersensitivity response to recall antigens and to the pseudomonas vaccine, and quantification of peripheral blood T and B cell levels and of in vitro lymphocyte blastogenic responses to commonly used mitogens and pseudomonas vaccine failed to demonstrate significant change from pretreatment values. Clinical trials to evaluate the therapeutic efficacy of pseudomonas vaccine with or without chemotherapy can be undertaken safely with this route and schedule.     

Visualization of mosaicism in tissues of normal and mismatch-repair-deficient mice carrying a microsatellite-containing transgene

We tested for azoospermia factor (AZF) deletions 17 loci corresponding to AZF subintervals a-d in 17 cases of testicular tumors occurring in Finns. While DNA samples from 48 CEPH and 32 Finnish males showed no deletions, patients with testicular cancer displayed AZF deletion mosaicisms in various non-tumor tissues (13 cases) and specific deletion haplotypes in tumor tissues (10 cases). Two of the cases with AZF deletions were testicular non-Hodgkin lymphomas indicating that Y-microdeletions appear also in malignancies other than seminoma and non-seminoma tumors. In good agreement with this assumption, we detected one AZF deletion in normal cells from 1 of 5 HNPCC cases, heterozygous for an MLH1 mutation. We propose that AZF deletions occur in early embryogenesis due to mutations of TSPY, mismatch repair (MMR), or X-specific genes. Since fathers of testicular, tumor cases did not exhibit AZF deletions, we assumed they were not carriers of the mutation inducing AZF deletion-mosaicisms. Therefore, tumor cases should have received the MMR gene or X mutations via the maternal lineage, or for the case of TSPY and MMR genes via a sperm carrying a mutation occurred in the paternal germ-cell line. We consider AZF microdeletions in non-tumor cells to be part of a broader pattern of chromosome instability producing susceptibility to testicular tumors. Clonal transformation and expansion of one of these tumor-susceptible cell lineages give rise to testicular tumors showing genome anomalies characteristic of testicular cancers (i12p, LOH and genetic imbalance for various autosomal regions, Y- and autosomal MSI, specific AZF deletion haplotypes).     

Blocking of lymphokine activated killer (LAK) cell mediated cytotoxicity by cell-sized beads bearing tumor cell proteins

Lymphokine activated killer cells (LAK) have been demonstrated to be cytotoxic for a variety of tumor-derived cells. Little is known of the nature of the cell surface molecules that mediate LAK cell-target cell interactions. Reported here are studies designed to develop the methodology that can lead to the identification and characterization of tumor cell surface molecules recognized by LAK cells. Results from experiments involving the pre-treatment of LAK cells and target cells (51Cr-labeled target cells or cold-blocking cells) with trypsin, neuraminidase, or sodium periodate suggest that proteins on the surface of LAK cells specifically recognized trypsin-sensitive molecules on the tumor cell surface. We extracted tumor cell membranes with detergents, and incorporated membrane proteins together with phospholipids and cholesterol onto the surfaces of cell-sized hydrophobic beads. The resulting "pseudocytes" block LAK mediated killing of 51Cr-labeled targets. Trypsin pretreatment of these pseudocytes significantly reduced their blocking activity. These observations suggested that we have incorporated onto the surface of pseudocytes tumor-membrane derived molecules that are specifically recognized by LAK cells. When membrane proteins from LAK resistant PBMC were incorporated onto beads, the resulting pseudocytes did not block LAK mediated cytotoxicity. It is of interest that beads coated with membrane proteins from one tumor were able to reduce LAK cell lysis of a different tumor target. Our results are consistent with the possibility that each LAK cell is polyspecific or that the LAK cell recognizes a common marker on many tumors. The methodology using pseudocytes should allow the purification and characterization of target acceptor molecule(s) and permit us to distinguish between these possibilities.     

Evaluation of biomedical text-mining systems: lessons learned from information retrieval

Biomedical text-mining systems have great promise for improving the efficiency and productivity of biomedical researchers. However, such systems are still not in routine use. One impediment to their development is the lack of systematic and rigorous evaluation, comparable to the approaches developed for information retrieval systems. The developers of text-mining systems need to improve both test collections for system-oriented evaluation and undertake user-oriented evaluations to determine the most effective use of their systems for their intended audience.     

Susceptibility of amyloid beta peptide degrading enzymes to oxidative damage: a potential Alzheimer's disease spiral

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.     

A clinical and molecular study of mosaicism for trisomy 17

Trisomy 17 has never been reported in a live birth. We present a case of mosaic trisomy 17 in a male presenting with mental retardation, seizures, attention deficit hyperactivity and autistic disorders, hearing loss, growth retardation, microcephaly, and minor anomalies. Although peripheral blood lymphocyte chromosomes were normal, trisomy 17 was present in the skin fibroblasts. The percentage of abnormal cells appears to have increased from 18% in an initial skin biopsy at age 3 years 8 months to 80% at age 8 years 8 months. Molecular analysis using 13 highly polymorphic markers spanning the length of chromosome 17 demonstrated the extra chromosome 17 in the skin to be of paternal origin. Three alleles were never seen in the trisomic cell line, suggesting that the extra chromosome arose through a mitotic duplication error after conception. Uniparental disomy was excluded in the euploid blood sample. Although Smith-Magenis syndrome involves a deletion of proximal 17p, some of the clinical features of this mosaic trisomy 17 patient, such as decreased REM sleep and increased tolerance to pain, are suggestive of phenotypic features observed in Smith-Magenis syndrome. We speculate that there are dosage-sensitive genes located in 17p11.2 that produce these phenotypes for either deficiencies or overexpression of their gene products.     

Reaction of neutral endopeptidase 24.11 (enkephalinase) with arginine reagents

The effect of the arginine-specific reagents phenylglyoxal and butanedione on the activity of neutral endopeptidase 24.11 ("enkephalinase") was determined. Inactivation of the enzyme by butanedione is completely protected by methionine-enkephalin, but only partially protected by methionine-enkephalinamide. In contrast, phenylglyoxal inactivation of the enzyme exhibits saturation kinetics with a Kd of 20 mM. The enzyme is only partially protected against phenylglyoxal inactivation by both methionine-enkephalin and its amide, indicating that phenylglyoxal reacts at two sites. Reaction of the enzyme with phenylglyoxal in the presence of saturating methionine-enkephalin involves the direct reaction of the reagent with the enzyme-substrate complex. Enzyme treated with butanedione or with phenylglyoxal (at site 1) exhibits a 3-5 decrease in substrate binding with little change in kcat. In contrast, reaction with phenylglyoxal in the presence of saturating methionine-enkephalin shows little change in substrate binding but a 4-fold decrease in kcat. Enzyme inactivation involves the incorporation of approximately 1 mol of phenylglyoxal/enzyme subunit in the absence of methionine-enkephalin and approximately 2.5 mol of phenylglyoxal/enzyme subunit in the presence of saturating methionine-enkephalin. These results suggest that an arginine residue on the enzyme is involved in substrate binding.