Lack of UDS activity in the livers of rats exposed to allylisothiocyanate

Drug-metabolizing cytochrome P450 enzymes, the major phase I enzymes, are active in human liver already at very early stages of intrauterine development, although presumably at fairly low concentrations and in low numbers. During maturation, these enzymes go through various developmental programmes towards adulthood. The major increase both in abundance as well as in number of different enzymes takes place after birth, probably during the first year of life. Detailed information concerning these developmental changes is still limited. The major drug-metabolizing P450 enzymes appear to be primarily members of the CYP3A subfamily in all stages of development. The balance between different members of this subfamily, however, undergoes significant switches from the foetal predominant CYP3A7 to the major adult form CYP3A4. The ontogeny of the other cytochrome P450 enzymes is less well characterized, but the major switch-on appears to occur mainly after birth. Developmental expression of P450 enzymes is one of the key factors determining the pharmacokinetic status of developing individuals both pre- and postnatally.     

DNA repair functions in heterologous cells

Our genetic information is constantly challenged by exposure to endogenous and exogenous DNA-damaging agents, by DNA polymerase errors, and thereby inherent instability of the DNA molecule itself. The integrity of our genetic information is maintained by numerous DNA repair pathways, and the importance of these pathways is underscored by their remarkable structural and functional conservation across the evolutionary spectrum. Because of the highly conserved nature of DNA repair, the enzymes involved in this crucial function are often able to function in heterologous cells; as an example, the E. coli Ada DNA repair methyltransferase functions efficiently in yeast, in cultured rodent and human cells, in transgenic mice, and in ex vivo-modified mouse bone marrow cells. The heterologous expression of DNA repair functions has not only been used as a powerful cloning strategy, but also for the exploration of the biological and biochemical features of numerous enzymes involved in DNA repair pathways. In this review we highlight examples where the expression of DNA repair enzymes in heterologous cells was used to address fundamental questions about DNA repair processes in many different organisms.     

Digestion in the newborn

Although the various aspects of digestion in the newborn have been studied for decades, we still lack quantitative information about the contribution of individual enzymes to the overall process. The information to date indicates that in spite of immaturity of many of the classical digestive mechanisms of the adult, the infant uses a number of compensatory systems to achieve adequate digestion of nutrients (Fig. 1). Thus, whereas in the infant gastric proteolysis is probably extremely limited, intestinal protein digestion is adequate. Although starch supplements are better tolerated in breast-fed infants, because of the compensation provided by human milk amylase, the infant is able to digest lactose and short-chain glucose polymers with endogenous brush border enzymes. Fat digestion is markedly aided by gastric lipase and, in breast-fed infants, the bile salt-dependent lipase of human milk. Thus, in the infant, gastric lipolysis is quantitatively much more significant than in adults. The absorption of human milk whey proteins (and probably also cow milk proteins) is probably associated more with the highly glycosylated form of these proteins than with immaturity of neonatal digestive enzymes.     

Responses to vaginal birth after cesarean section

Recently, significant new insight has been obtained into the structure and catalytic mechanism of enzymes that convert environmental pollutants. Recent advances in protein engineering make it possible to use this information for improving the catalytic performance of such enzymes to achieve increased stability and expanded substrate range.     

Structural biology of HIV

The human immunodeficiency virus (HIV) genome encodes a total of three structural proteins, two envelope proteins, three enzymes, and six accessory proteins. Studies over the past ten years have provided high-resolution three-dimensional structural information for all of the viral enzymes, structural proteins and envelope proteins, as well as for three of the accessory proteins. In some cases it has been possible to solve the structures of the intact, native proteins, but in most cases structural data were obtained for isolated protein domains, peptidic fragments, or mutants. Peptide complexes with two regulatory RNA fragments and a protein complex with an RNA recognition/encapsidation element have also been structurally characterized. This article summarizes the high-resolution structural information that is currently available for HIV proteins and reviews current structure-function and structure-biological relationships.     

Developing nurse practitioners to develop practice: the experiences of nurses working on a nursing development unit

Drug metabolism influences the pharmacotoxicological properties of a vast array of compounds and is controlled by a complex system of drug-metabolizing enzymes. A thorough understanding of this system allows the more effective development of therapeutic drugs as well as a significant improvement of risk assessment. The early identification of optimal therapeutic problems relating to drug metabolism could reduce the development costs for pharmaceuticals. Recently, techniques using transgenes have become available for this purpose. In these approaches the genetic information for the enzyme under investigation is expressed in vitro or in vivo, following gene transfer. This approach is called 'heterologous expression'. This article illustrates some examples in which molecular biological methods have been used to analyze those enzymes which control the pharmacotoxicological properties of drugs. Particular emphasis has been placed on the use of these methods to characterize extrahepatic drug-metabolizing enzymes such as those in the skin.     

The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases

This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products. The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program. These artificial substrates have been widely used to produce chiral synthons. Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates. Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.     

Structures and mechanisms of glycosyl hydrolases

The wealth of information provided by the recent structure determinations of many different glycosyl hydrolases shows that the substrate specificity and the mode of action of these enzymes are governed by exquisite details of their three-dimensional structures rather than by their global fold.     

Organization of the human SP-A and SP-D loci at 10q22-q23. Physical and radiation hybrid mapping reveal gene order and orientation

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15-18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed.     

Recent advances in the biology of central nervous system tumors

BACKGROUND: In order to study the biosynthesis of vitamin B12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. RESULTS: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-L-methionine were included in the incubation, the product was precorrin-4. Both precorrin 3B and precorrin-4 are known precursors of vitamin B12 and their availability has allowed new mechanistic studies of enzymic transformations. CONCLUSIONS: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B12 biosynthesis.     

Proline specific peptidases

Proline is unique among the 20 amino acids due to its cyclic structure. This specific conformation imposes many restrictions on the structural aspects of peptides and proteins and confers particular biological properties upon a wide range of physiologically important biomolecules. In order to adequately deal with such peptides, nature has developed a group of enzymes that recognise this residue specifically. These peptidases cover practically all situations where a proline residue might occur in a potential substrate. In this paper we endeavour to discuss these enzymes, particularly those responsible for peptide or protein hydrolysis at proline sites. We have detailed their discovery, biochemical attributes and substrate specificities and have provided information as to the methodology used to detect and manipulate their activities. We have also described the roles, or potential roles that these enzymes may play physiologically and the consequences of their dysfunction in varied disease states.     

Gamma-irradiation and UV-C light-induced lipid peroxidation: a Fourier transform-infrared absorption spectroscopic study

Metabolism of most drugs influences their pharmacological and toxicological effects. Drugs particularly affected are those with a narrow therapeutic window and that are subjected to considerable first-pass metabolism. Much of the interindividual and interethnic differences in effects of drugs is now attributable to genetic differences in their metabolism. Genetic polymorphisms have been described for many drug-metabolising enzymes in Caucasian and Oriental populations, the most well-characterised being those for cytochrome P450 2D6, cytochrome P450 2C19, glutathione S-transferases, and N-acetyl transferase 2. African populations have been studied to a lesser extent, but it is apparent that populations within Africa are heterogeneous with respect to these polymorphisms. In addition, although some allelic variants are common to all populations throughout the world (e.g., CYP2D6*5), some allelic variants are specific for an African population (e.g., CYP2D6*17). The polymorphisms give rise to enzymes with changed or no activity towards drug substrates. Two of the most important enzymes for metabolism of neuroleptics and other psychoactive drugs are CYP2D6 and CYP2C19. This article compares the current information on polymorphisms of these two enzymes in African and other populations and discusses the implications of these polymorphisms for neuropharmacotherapy.     

Biodegradation of atrazine under denitrifying conditions

Several significant advances in the understanding of the catalytic mechanisms, structures and evolution of glutathione transferases have occurred in the past year. These advances include new mechanistic information concerning the canonical soluble enzymes, the finding that the fosfomycin-specific enzyme, FosA, is a metalloglutathione transferase and a higher resolution projection structure of the microsomal enzyme.     

Extremozymes: expanding the limits of biocatalysis

The study of enzymes isolated from organisms inhabiting unconventional ecosystems has led to the realization that biocatalysis need not be constrained to mild conditions and can be considered at pH's, temperatures, pressures, ionic and solvent environments long thought to be destructive to biomolecules. Parallel to this, it has been demonstrated that even conventional enzymes will catalyze reactions in solvents other than water. However, the intrinsic basis for biological function under extreme conditions is only starting to be addressed, as are associated applications. This was the focus of a recent NSF/NIST-sponsored workshop on extremozymes. Given the information acquired from the study of extremozymes, modification of enzymes to improve their ranges of stability and activity remains a possibility. Ultimately, by expanding the range of conditions suitable for enzyme function, new opportunities to use biocatalysis will be created.     

Thymidine kinase mutants obtained by random sequence selection

Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.     

The molecular basis of quantitative genetic variation in central and secondary metabolism in Arabidopsis

To find the genes controlling quantitative variation, we need model systems where functional information on physiology, development, and gene regulation can guide evolutionary inferences. We mapped quantitative trait loci (QTLs) influencing quantitative levels of enzyme activity in primary and secondary metabolism in Arabidopsis. All 10 enzymes showed highly significant quantitative genetic variation. Strong positive genetic correlations were found among activity levels of 5 glycolytic enzymes, PGI, PGM, GPD, FBP, and G6P, suggesting that enzymes with closely related metabolic functions are coregulated. Significant QTLs were found influencing activity of most enzymes. Some enzyme activity QTLs mapped very close to known enzyme-encoding loci (e.g., hexokinase, PGI, and PGM). A hexokinase QTL is attributable to cis-acting regulatory variation at the AtHXK1 locus or a closely linked regulatory locus, rather than polypeptide sequence differences. We also found a QTL on chromosome IV that may be a joint regulator of GPD, PGI, and G6P activity. In addition, a QTL affecting PGM activity maps within 700 kb of the PGM-encoding locus. This QTL is predicted to alter starch biosynthesis by 3.4%, corresponding with theoretical models, suggesting that QTLs reflect pleiotropic effects of mutant alleles.     

Synthesis and applications for unnatural sugar nucleotides

The synthesis and biological evaluation of carbohydrate mimetics has begun to more clearly define the diverse roles of carbohydrates in nature. Often the strategy invoves the design and synthesis of glycosyltransferase and glycosidase inhibitors both as tools to elucidate the mechanism of action of these enzymes and as potential therapeutic agents. An array of unnatural sugar nucleotides have found utility in chemo-enzymatic synthesis. The regio- and stereoselective transfer of sugars by glycosyltransferases such as b1,4-galactosyltransferase, a1,3-fucosyltransferase, a2,3- and a2, 6-sialyltransferases and N-acethylglucosaminyltransferase V has demonstrated the broad application of this approach. This review summarizes the specificity of these well-studied glycosyltransferases for both unnatural sugar donors and acceptors. This information combined with the knowledge of the mechanism of action of those enzymes is valuable in the design of potent selective glycosyltransferase inhibitors and the chemo-enzymatic synthesis of novel carbohydrate mimetics.     

Molecular stereopharmacology and the search for stereoselective drugs

Analyzing modern trends in stereopharmacology shows that its theoretical aspects are mainly associated with the studies of the steric parameters of active centers of drug receptors and enzymes by using compounds having a well-established stereostructure. Applied stereopharmacology underlines the vital necessity of selective production and use of drug eutomers and active modifications of polymorphic drugs. The creation of an information computer database of stereostructural parameters of drugs is considered to be basic for further design of new selective drugs.     

Is there an error correcting code in the base sequence in DNA?

Modern methods of encoding information into digital form include error check digits that are functions of the other information digits. When digital information is transmitted, the values of the error check digits can be computed from the information digits to determine whether the information has been received accurately. These error correcting codes make it possible to detect and correct common errors in transmission. The sequence of bases in DNA is also a digital code consisting of four symbols: A, C, G, and T. Does DNA also contain an error correcting code? Such a code would allow repair enzymes to protect the fidelity of nonreplicating DNA and increase the accuracy of replication. If a linear block error correcting code is present in DNA then some bases would be a linear function of the other bases in each set of bases. We developed an efficient procedure to determine whether such an error correcting code is present in the base sequence. We illustrate the use of this procedure by using it to analyze the lac operon and the gene for cytochrome c. These genes do not appear to contain such a simple error correcting code.