An enzyme electrode for acetylcholine.

A new enzyme electrode is described to measure continuously acetylcholine concentration. A coating containing active acetylcholinesterase is produced on a pH-glass electrode. The mean thickness of the coating is 50 micrometer. Optimal operational conditions with respect to buffer concentration, ionic strength, linearity, stability, sensitivity, pH of the bulk solution, and response time are studied and discussed. The use of acetylcholinesterase-containing membranes as sensors could offer several novel advantages.     

Influence of cholesterol status on blood lipid and lipoprotein enzyme responses to aerobic exercise.

To compare postexercise changes in plasma lipids and lipoprotein enzymes in 13 hypercholesterolemic (HC) and 12 normocholesterolemic men [total cholesterol (TC) 252 +/- 5 vs. 179 +/- 5 mg/dl], fasting blood samples were obtained 24 h before, immediately, 24, and 48 h after a single bout of treadmill walking (70% peak O(2) consumption, 500 kcal expenditure). Significant findings (P < 0.05 for all) for plasma volume-adjusted lipid and enzyme variables were that TC, low-density-lipoprotein cholesterol, and cholesterol ester transfer protein activity were higher in the HC group but did not influence the lipid responses to exercise. Across groups, TC was transiently reduced immediately after exercise but returned to baseline levels by 24 h postexercise. Decreases in triglyceride and increases in high-density-lipoprotein cholesterol (HDL-C) and HDL(3)-C were observed 24 h after exercise and lasted through 48 h. Lipoprotein lipase activity was elevated by 24 h and remained elevated 48 h after exercise. HDL(2)-C, cholesterol ester transfer protein activity, hepatic triglyceride lipase, and lecithin: cholesterol acyltransferase activities did not change after exercise. These data indicate that the exercise-induced changes in HDL-C and triglyceride are similar in HC and normocholesterolemic men and may be mediated, at least in part, by an increase in lipoprotein lipase activity.     

Structural basis for broad substrate specificity of earthworm fibrinolytic enzyme component A

Earthworm fibrinolytic enzyme component A (EFE-a) possesses an S1 pocket, which is typical for an elastase-like enzyme, but it can still hydrolyze varieties of substrates, and it exhibits wide substrate specificity. Former structure studies suggested that the four-residue insertion after Val(217) might endow EFE-a with this specificity. Based on the native crystal structure at a resolution of 2.3A, we improved the native crystal structure to 1.8A and determined its complex structure with the inhibitor Meo-Suc-Ala-Ala-Pro-Val-CMK at a resolution of 1.9A. The final structures show that: (1) EFE-a possesses multisubstrate-binding sites interacting with the substrates; (2) significant conformation adjustment takes place at two loops binding to the N-terminal of the substrates, which may enhance the interaction between the enzyme and the substrates. These characteristics make the substrate-specificity of EFE-a less dependent on the property of its S1-pocket and may endow the enzyme with the ability to hydrolyze chymotrypsin-specific substrates and even trypsin-specific substrates.     

Series mechanism of enzyme deactivation. Characterization of intermediate forms

Acid phosphatase (E.C. 3.1.3.2) undergoes complex thermal deactivation phenomena, as revealed by the two-slope pattern of the enzyme logarithmic-specific-activity versus time curves. The native enzyme first decays toward an equilibrium distribution of less, but still active, intermediate structures and these, in turn, undergo a final degradation to a completely inactive form. The effect of the experimental conditions at which the enzyme is kept during the deactivation process on the characteristics of these intermediate enzymatic structures has been investigated. The kinetic parameters of p-nitro-phenyl phosphate hydrolysis, as catalyzed by some of these intermediate forms, have been determined and the results compared to those obtained with the native enzyme.     

The distribution of keratin type intermediate filaments in human breast cancer. An immunohistological study

Antibodies to different intermediate filament proteins can be used to distinguish cells of epithelial, mesenchymal, muscle, glial and neuronal origin. Antibodies to prekeratin which characterize cells of epithelial origin, and antibodies to vimentin which recognize cells of mesenchymal origin have been used to study twenty cases of breast carcinoma (sixteen infiltrating ductal carcinomas and four infiltrating intraductal carcinomas), two cases of cystic breast disease, two fibroadenomas and one case of benign cystosarcoma phylloides. The prekeratin and vimentin were detected using specific antibodies to these proteins by immunofluorescence microscopy using alcohol fixed paraffin-embedded tissues. In eighteen out of the twenty carcinomas the tumor cells were strongly and specifically stained by antibodies to prekeratin. DIfferent tumors gave different patterns of prekeratin staining. In contrast, when the same specimens were tested with the vimentin antibody, the tumor cells were unstained, and instead only the usual strong staining to fibroblasts and blood vessels in the stroma was observed. In cystic breast disease, fibroadenomas, and benign cystosarcoma phylloides, cells of epithelial origin were strongly stained by the prekeratin but not by the vimentin antibody.     

A genetic basis for metastasis.

The progression of a normal cell to one that is malignant is characterized by at least four progressive but potentially separable behavioural patterns identifying the immortal, tumorigenic, invasive and metastatic phenotypes. A multitude of steps appear to be involved in both transformation from normality and progression to malignancy as characterized by the acquisition of metastatic behaviour. Consequently, it seems unlikely that a single gene can directly manifest expression of the metastatic phenotype in normal cells unless it can induced pleiotropic effects. Indeed, a single trait uniquely characterizing the metastatic phenotype has never been identified. The possibility of a single gene suppressing the metastatic phenotype seems much greater. One possible candidate for such a gene is nm23, the expression of which correlates with reduced metastatic potential in several tumours including breast cancer in humans. Although the numbers involved are still small, the correlation of nm23 expression with breast cancer outcome offers potential in using this system as a prognostic aid in clinical diagnosis of this disease. Its possible role as an indicator of metastatic potential in other human tumours remains to be evaluated, although current evidence suggests that it is unlikely to be of use in colon cancer. Further significant progress requires molecular dissection of the mode of action of its product.     

An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis.

The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.     

A novel type of deubiquitinating enzyme

A previous report from this laboratory described two novel proteins that have sequence similarity to A20, a negative regulator of NF-kappaB (Evans, P. C., Taylor, E. R., Coadwell, J., Heyninck, K., Beyaert, R., and Kilshaw, P. J. (2001) Biochem. J. 357, 617-623). One of these molecules, cellular zinc finger anti-NF-kappaB (Cezanne), a 100-kDa cytoplasmic protein, also suppressed NF-kappaB. Here we demonstrate that Cezanne is a novel deubiquitinating enzyme, distinct from the two known families of deubiquitinases, Types I and II. We show that Cezanne contains an N-terminal catalytic domain that belongs to the recently discovered ovarian tumor protein (OTU) superfamily, a group of proteins displaying structural similarity to cysteine proteases but having no previously described function. Recombinant Cezanne cleaved ubiquitin monomers from linear or branched synthetic ubiquitin chains and from ubiquitinated proteins. Mutation of a conserved cysteine residue in the catalytic site of the proteolytic domain caused Cezanne to co-precipitate polyubiquitinated cellular proteins. We also provide evidence for an additional ubiquitin binding site in the C-terminal part of the molecule. Our data provide the first demonstration of functional activity among OTU proteins.     

An anatomical basis for prognosis of malignant melanoma.

Clinical observations during surgery have led us to speculate that an anatomic basis may in fact exist for the poor prognosis associated with malignant melanoma arising in certain recognized "high-risk" areas. In our study we have treated and followed 45 patients with primary malignant melanoma for over 5 years. During the course of treatment, we identified variations in anatomic characteristics at the tumor sites. Criteria were established for high- and low-risk locations by the neurovascular structure encountered. We speculate that these sites that have neurovascular windows provide a readily accessible vascular pathway for the dissemination of malignant cells to deeper visceral structures and may account for the poor prognosis associated with primary lesions in these locations. Thirty patients were classified as being at high risk for developing metastasis, whereas 13 were classified as being at low risk; 2 patients were in a special-risk category. To date, 12 of the 30 patients with "high-risk" melanoma have gone on to develop metastatic disease, which represents 40 percent of that group, whereas none of the patients classified as "low risk" have developed metastases during the same period.     

Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme.

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.     

A molecular basis for auxin action.

The plant hormone auxin is central in the regulation of growth and development, however, the molecular basis for its action has remained enigmatic. In the absence of a molecular model, the wide range of responses elicited by auxin have been difficult to explain. Recent advances using molecular genetic approaches in Arabidopsis have led to the isolation of a number of key genes involved in auxin action. Of particular importance are genes involved in channelling polar auxin transport through the plant. In addition a model for auxin signal transduction, centred on regulated protein degradation, has been developed.