Quantitative Histochemistry: Histologic Sampling of Sections of Frozen-Dried Tissues

Microchemical techniques are available for analyses of many enzymes and other components of tissue for which stains are not available. This paper describes techniques of freeze drying and histological sampling of certain cell groups or structures free from surrounding stroma, fat and other types of cells. Quantitative data can be obtained for many enzymes in 0.5 to 5 μ samples of freeze-dried tissue. For example, glucoses-phosphate dehydrogenase activity was measured and compared in the breast alveoli of mice in estrus, diestrus, gestation and lactation.     

A Pre-embedding Reaction Method for Localizing NADH Reductase and Succinic Dehydrogenase in Skeletal Muscle

Paraffin wax embedding methods suitable for demonstrating the distribution of enzyme activity in tissues sections are uncommon; most procedures rely on the use of frozen section techniques. This paper describes a system for demonstrating certain enzymes which involves incubation of the tissue with appropriate substrates before a Paramat wax embedding procedure. While it has distinct merits of its own, the procedure is eminently suitable for use where a cryostat is not available; it can also be readily applied to other enzymes and tissues.     

Use of a restriction enzyme-digested PCR product as substrate for helicase assays

DNA helicases play essential roles in many cellular processes. The currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. Here, a PCR-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated.     

Applications of recombinant DNA technology to the pulp and paper industry

New gene selection techniques (Recombinant DNA) are currently available to exploit useful properties of various biological systems hitherto regarded as interesting but of little or no immediate commercial value. The application of genetic engineering techniques to problems in the Pulp and Paper Industry are many. As a first step these techniques are being used to provide much needed fundamental information on the cellular and molecular mechanisms involved in the expression of extra-cellular enzymes that degrade lignocellulosic pulping wastes. The information gleaned from the studies on cellulolytic fungi and bacteria can be used to genetically engineer a yeast or bacterium capable of converting pulping wastes into ethanol and other useful by-products.     

Polysaccharases for microbial exopolysaccharides

Microbial exopolysaccharides (EPS) are the substrates for a wide range of enzymes most of which are highly specific. The enzymes are either endoglycanases or polysaccharide lyases and their specificity is determined by carbohydrate structure with uronic acids often playing a major role. The presence of various acyl substituents frequently has little effect on the action of many of the polysaccharases but markedly inhibits some of the polysaccharide lyases including alginate and gellan lyases. The commonest sources of such enzymes can be either microorganisms or bacteriophages. These specific polysaccharide-degrading enzymes can yield oligosaccharide fragments, which are amenable to NMR and other analytical techniques. They have thus proved to be extremely useful in providing information about microbial polysaccharide structures and were routinely used in many such studies. Complex systems containing various mixtures of enzymes may also be effective in the absence of single enzymes but may be difficult to obtain with reproducible activities. Such preparations may also cause extensive degradation of the polysaccharide structure and thus prove less useful in providing information. Commercially available enzyme preparations have seldom proved capable of degrading microbial heteropolysaccharides, although some are active against bacterial alginates and homopolysaccharides including bacterial cellulose and curdlan.     

Amino acid biosynthesis in plants: Approaching an understanding at the molecular level

Summary The genes encoding GS and EPSP synthase have already been cloned. Clones containing portions of the genes encoding aspartate amino transferase and asparagine synthetase tentatively have been identified by this and other laboratories. It is certain that many other genes encoding other important enzymes involved in amino acid biosynthesis will also be identified in the near future. It is evident that new techniques for the separation of proteins will greatly aid in identifying low abundance genes more rapidly and efficiently. Micro techniques need to be further developed and the necessary equipment made available to laboratories. Hopefully the cost of the required equipment will decrease and commercial enterprises will offer more contract services for work requiring the more expensive equipment. Graduate training in advanced techniques of separating proteins at high resolutions should be encour-aged expanded so that our future scientists are well versed in protein biochemistry as well as in techniques of molecular biology.     

Specific {beta} glucanases as tools for polysaccharide structure determination

A number of interesting ß-glucanases with a varietyof substrate specificities have recently been described. Theseenzymes are ideal tools for elucidating the structure of thewide array of glucose polymers found in nature. Since many ofthe enzymes can be easily produced in substantial quantitiesby cloning and ‘overexpression’ techniques, theyare available for other purposes as well. For example, theymay substantially increase the nutritional value of grain andother plant materials. glucanases ß-glucans polysaccharides     

Articular cartilage and changes in Arthritis: Matrix degradation

While many proteases in articular cartilage have been described, current studies indicate that members of two families of metalloproteases – MMPs and the ADAMTSs – are responsible for the degradation of the major components of this tissue. Collagenases (MMPs) make the first cleavage in triple-helical collagen, allowing its further degradation by other proteases. Aggrecanases (ADAMTSs), in conjunction with other MMPs, degrade aggrecan, a component of the proteoglycan aggregate. Anti-neoepitope antibodies that recognize the cleavage products of collagen and aggrecan generated by these enzymes are now available and are being used to detect the sites of action and to quantitate degradation products.     

Simultaneous preparation from human placenta of several enzymes of glucose metabolism

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.     

Diazonium inactivation in simultaneous-coupling and product inhibition in post-coupling azo-techniques for demonstrating activity of acid hydrolases

In this communication results are presented of an investigation in which the activity of the hydrolytic enzymes acid phosphatase, beta-glucuronidase, non specific arylesterase, microsomal arylsulphatase, beta-galactosidase, beta-N-acetylglucosaminidase, acid alpha-glucosidase and aminopeptidase M are demonstrated in tissue sections with simultaneous- and post-coupling azo-techniques. Semipermeable membrane techniques are used to hamper enzyme diffusion during the incubation period. From the histochemical and biochemical findings it appeared that an advantage of the post-coupling techniques over the simultaneous-coupling techniques is that inactivation of the enzymes by the coupling reagents is avoided. On the other hand post-coupling techniques are subject to product inhibition. With kinetic inhibition studies it is found that for microsomal arylsulphatase and non-specific arylesterase this product inhibition is non-competitive. This product inhibition may be a problem for histochemical quantitative post-coupling techniques for the determination of acid hydrolase activity.     

Strategy and success for the directed evolution of enzymes

Directed evolution is widely used to improve enzymes, particularly for industrial biocatalytic processes. Molecular biology advances present many new strategies for directed evolution. Commonly used techniques have led to many successful examples of enzyme improvement, yet there is still a need to improve both the efficiency and capability of directed evolution. Recent strategies aimed at making directed evolution faster and more efficient take better advantage of available structural and sequence information. The underlying principles that lead to early dead-ends for directed evolution experiments are also discussed along with recent strategies designed to by-pass them. Several emerging methods for creating novel enzymes are also discussed including examples of catalytic activity for which there is no precedent in nature. Finally, the combined use of several strategies is likely to be required in practice to improve multiple target properties of an enzyme, as successfully shown by a recent industrial example.     

Controls of citrate synthase activity

The inhibition of citrate synthase by a variety of nucleotides and polycarboxylate compounds is not unexpected since many of the compounds are substrate analogs of citrate synthase. These effectors are interesting by virtue of the fact that many of them are intermediates and/or end products in the metabolic path of which citrate synthase can be considered the first committed step. As a consequence, it is possible to propose regulation of citrate synthase by ATP (or phosphorylation potential) by acyl CoA (acylation level) and NADH (redox potential). Aside from these putative controls, it is possible that the major control of citrate synthase activity is by changes in the concentration of its substrates acetyl CoA and oxalacetate.I discuss in this review the many factors that must be considered before one can decide whether or not interactions between metabolites and enzymes observed in an $\text{in vitro}$ catalytic situation have metabolic relevance. These factors include 1) the concentrations of substrates at the enzyme site, 2) the concentrations of effectors at the enzyme site, 3) the presence of modifying substances, and 4) the difference in behavior of an enzyme at its concentration $\text{in vivo}$ compared to its concentration $\text{in vitro}$. In the case of citrate synthase as is generally true for other enzymes, no accurate knowledge of these factors are available $\text{in vitro}$ so that little can be said concerning the $\text{in situ}$ control of citrate synthase, which may be the result of all the factors acting in concert. The studies of effectors on enzymes $\text{in vitro}$ can only serve as a guideline for parameters to study when techniques are available to study control of enzymes $\text{in situ}$.     

Fungal biotechnology

Fungi are used in many industrial processes, such as the production of enzymes, vitamins, polysaccharides, polyhydric alcohols, pigments, lipids, and glycolipids. Some of these products are produced commercially while others are potentially valuable in biotechnology. Fungal secondary metabolites are extremely important to our health and nutrition and have tremendous economic impact. In addition to the multiple reaction sequences of fermentations, fungi are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine-chemical industry in the production of single-isomer intermediates. Recombinant DNA technology, which includes yeasts and other fungi as hosts, has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as a means of increasing titers and yields of microbial processes and in the discovery of new drugs. Today, fungal biology is a major participant in global industry. Moreover, the best is yet to come as genomes of additional species are sequenced at some level (cDNA, complete genomes, expressed sequence tags) and gene and protein arrays become available.     

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.     

Dipeptidase-C inDrosophila melanogaster: Genetic,ontogenetic, and tissue-specific variation

Dip-A, Dip-B, and Dip-C constitute structural genes for three peptidic enzymes in Drosophila melanogaster distinct from the leucine aminopeptidases. Their ontogenetic and tissue distributions of activities suggest the involvement of these enzymes in a general metabolic role, such as the regulation of amino acid and oligopeptide pools to make amino acids available for protein synthesis. Screening of chromosome substitution isogenic lines for DIP-C activity indicated that, like DIP-A and DIP-B, unlinked activity modifiers exist for Dip-C. The developmental profiles of dipeptidase activities are very similar, except in the pupal stage, during which DIP-C activity is markedly low compared to the other two enzymes. Intercorrelations of dipeptidase activities vary ontogenetically, which is consistent with the need for coordinate expression of these enzymes during certain developmental stages. Tissue-specific expression of dipeptidases in larvae and adults are also similar, although the relative levels of DIP-A activity differ from those of DIP-B and DIP-C in certain organs and body parts. Some of the differences among chromosome substitution lines for dipeptidase activities appear to be systemic, while others are developmental stage-specific and tissue-specific. Second- and third-chromosome variants for DIP-C activity differed in their tissue distribution. This is consistent with the presence of temporal and spatial variants in natural populations for other Drosophila enzymes.     

Mechanized techniques for the selective extraction of enzymes from plant epidermal glands

Many plant products are biosynthesized and accumulated in epidermal glands. For investigations on the metabolism of these compounds it is most convenient to obtain cell-free preparations enriched in gland contents. Two simple mechanized procedures have been developed for gently abrading the plant surface in order to efficiently extract glandular enzymes in high purity. These methods allow rapid processing of large quantities of plant material and yield extracts largely uncontaminated with materials from underlying tissue. The use of these procedures for isolating several enzymes of terpenoid metabolism is described. These techniques work especially well for microsomal enzymes and may be useful not only for enzymes found in epidermal glands but also for other enzymes localized in or near the epidermis. With simple modification, these procedures can be adapted for use with a variety of different types of plant tissues.     

In situ sites for xenobiotic activation and detoxication: implications for the differential susceptibility of cells to the toxic actions of environmental chemicals

The findings reported in this communication illustrate that histochemical approaches can provide a significant amount of insight into an area of considerable toxicologic importance. Results of our immunohistochemical and histochemical studies clearly demonstrate that neither xenobiotic-metabolizing enzymes nor oxidative xenobiotic metabolism occur uniformly throughout tissues that often are damaged as a result of the bioactivation of environmental chemicals and other xenobiotics, that there can be significant differences in both the contents and activities of xenobiotic-metabolizing enzymes among even morphologically similar cells such as hepatocytes, and that enzyme inducers can alter differentially the extents to which different cells in a tissue metabolize xenobiotics. Knowledge of the precise intratissue localizations and distributions of xenobiotic-metabolizing enzymes and xenobiotic biotransformation reactions clearly is critical for defining the roles individual cells play in the metabolism of xenobiotics. It must be recognized, however, that the mere presence of xenobiotic-metabolizing enzymes in a cell cannot, by itself, explain why that cell might be highly susceptible to toxicities resulting from the bioactivation of certain xenobiotics. Thus, it is apparent that considerably more study is needed, especially in situ using histologic and cytologic techniques, in order to characterize the balance between xenobiotic activation and detoxication processes within individual cells in target tissues and elucidate the basis for the cell-selective nature of toxicities caused by the generation of reactive metabolites from many xenobiotics.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.