Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.     

Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests.     

BACE1 inhibitors: A head group scan on a series of amides

A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basicity as well as their properties as substrates of P-glycoprotein revealed that a 2-amino-1,3-oxazine head group would be a suitable starting point for further development of brain penetrating compounds for potential Alzheimer’s disease treatment.     

Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope,co-solvent tolerance and biocatalyst immobilization

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50?mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.     

Phosphonate utilization by bacteria.

Bacteria able to use at least one of 13 ionic alkylphosphonates of O-alkyl or O,O-dialkyl alkylphosphonates as phosphorus sources were isolated from sewage and soil. Four of these isolates used 2-aminoethylphosphonic acid (AEP) as a sole carbon, nitrogen, and phosphorus source. None of the other phosphonates served as a carbon source for the organisms. One isolate, identified as Pseudomonas putida, grew with AEP as its sole carbon, nitrogen, and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 72% of the AEP nitrogen as ammonium. This is the first demonstration of utilization of a phosphonoalkyl moiety as a sole carbon source. Cell-free extracts of P. putida contained an inducible enzyme system that required pyruvate and pyridoxal phosphate to release orthophosphate from AEP; acetaldehyde was tentatively identified as a second product. Phosphite inhibited the enzyme system.     

Biotechnological uses of cyanobacteria

Cyanobacteria (blue-green algae) are 0₂-evolving photosynthesizing prokaryotes that have an extensive history of use as a human food source and as a fertilizer in rice fields. They have also been recognized as an excellent source of vitamins and proteins and as such are found in health food stores in North America and elsewhere. Cyanobacteria have a great deal of potential as a source of fine chemicals, as a biofertilizer and as a source of renewable fuel. This potential is being realized as data from research in the areas of the physiology and chemistry of these organisms are gathered and as the knowledge of cyanobacterial genetics and genetic engineering increases. We review, here, the present (and possible future) uses of cyanobacteria and assess the state of the art with respect to the genetic manipulation of cyanobacteria.     

Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids

In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.     

The interaction of alpha-chlorohydrin with glycerol kinase.

alpha-Chlorohydrin has been examined both for its ability to act as a substrate for glycerol kinase and as an inhibitor of the reaction of glycerol with glycerol kinase. Using a purified enzyme from Candida mycoderma, it was established that alpha-chlorohydrin does not act as a substrate for glycerol kinase, but does act as a competitive inhibitor (Ki of 30 mM) of purified glycerol kinase and the enzyme present in a sonicated preparation of ram spermatozoa. Neither alpha-chlorohydrin nor alpha-chlorohydrin phosphate acted as inhibitors of NAD- or flavin-linked glycerolphosphate dehydrogenase. It is concluded that alpha-chlorohydrin does not cause the impairment of sperm metabolism as a result of phosphorylation catalysed by glycerol kinase.     

Nucleoside 5'-triphosphate analogs as positive and negative effectors of mammalian ribonucleotide reductase

Ribonucleotide reductase activity is strongly regulated by nucleoside 5'-triphosphates acting as positive and negative effectors. With the use of dGTP analogs, araGTP and dITP, it was found that the structural requirements of dGTP to serve as a positive effector of ADP reductase were not the same as the requirements for dGTP to serve as a negative effector of CDP and ADP reductase activities. The dTTP analogs methylenedTTP and dideoxyTTP also gave different responses in terms of activating GDP reductase activity and inhibiting CDP and ADP reductase activities. Etheno-ATP and etheno-dATP were inactive as positive and negative effectors, respectively, of CDP reductase activity. DideoxyATP was less active than dATP as a negative effector. Formycin ATP was a very poor substitute for ATP as a positive effector of CDP reductase. These studies indicate that the effector sites are very specific in terms of binding nucleoside triphosphates as positive or negative modulators of ribonucleotide reductase activity.     

Lanthanum as an electron microscopic stain

Applications of lanthanum as an electron microscopic tracer have been reviewed. This electron-dense trivalent cation, which binds avidly to calcium binding sites, can be used as tracer for delineating extracellular spaces and intercellular junctions. It has served as a basis for classification of junctional structures. It can also be used as a calcium probe, a tracer in studying the permeability of barriers, as an intracellular marker and as an electron microscopic stain for such membrane components as surface glycoprotein. Each of these applications may require a different methodology. Thus methodological considerations in the use of this tracer have also been reviewed. The recent recognition that lanthanum is more than a passive tracer and that by reacting with different cell components may serve as a true stain, will extend the use of lanthanum in electron microscope histochemistry.     

Fundamental-applied aspects of systemic inflammation in terms of physiologic and typical pathological process

The concept of systemic inflammatory response syndrome (SIRS) by sepsis as well as quality of SIRS criteria, classification, and PIRO system has been a subject to analytical criticism in terms of theory of physiologic and typical pathological process. It has been disclosed SIRS can be considered only as the syndrome, that solves particular clinical tasks, but not as a basic model of pathogenesis of critical states. In authors' opinion it is more correctly to discuss systemic inflammation as a typical pathologic process that appears as a complex of one or another "resuscitation" syndrome in a clinical course.     

Transforming growth factor beta1 and prostaglandin E2 concentrations are associated with bone formation markers in ulcerative colitis patients

Osteoporosis is a significant complication of a multifactoral etiology associated with inflammatory bowel disease. The aim of study was to evaluate the relationships between bone mineral density as well as bone turnover markers and inflammatory activity modulators (i.e., PGE2 and TGFbeta1) in ulcerative colitis (UC). Twenty-one active ulcerative colitis subjects and 14 healthy individuals were included into the study. We observed no significant differences in serum concentrations of osteoprotegerin and osteocalcin, as well as bone mineral density between UC patients and healthy individuals. Plasma concentrations of PGE2, TGFbeta1 and TNF-alpha were significantly higher in UC patients than in controls. Serum osteocalcin demonstrated a positive correlation with both serum PGE2 and plasma TGFbeta1. Moreover there was significant correlation between osteoprotegerin and TGFbeta1 as well as serum TNF-alpha concentrations. In conclusion a positive association between PGE2 and TGFbeta1 and bone formation markers-osteoprotegerin and osteocalcin, as well as a comparable BMD in UC patients and healthy individuals was shown. Our results may indicate that increase of PGE2 as well as TGFbeta1 concentrations may play a protective role against bone loss in ulcerative colitis patients.     

The cell as nexus: connections between the history, philosophy and science of cell biology

Although the cell is commonly addressed as the unit of life, historians and philosophers have devoted relatively little attention to this concept in comparison to other fundamental concepts of biology such as the gene or species. As a partial remedy to this neglect, we introduce the cell as a major point of connection between various disciplinary approaches, epistemic strategies, technological vectors and overarching biological processes such as metabolism, growth, reproduction and evolution. We suggest that the role of the cell as a nexus forms the basis for a new philosophical and historical appreciation of cell biology. This perspective focuses less on the cell as a well-defined, stable object and places more emphasis on its role as a mediator of fundamental biological processes.     

The Taxon as an Ontological Problem

Although the term taxon is one of the most common concepts in biology, a range of its meanings cannot be comprehended by an universal definition. Usually, biologists construe their knowledge of “the same” taxon by substantially different interpretations, so they find themselves in need either to justify this “multiplication of taxon essences”, or to surmount their plurality unifying its interpretations into a single explanation of what a taxon is. In both cases, an ontological status (“reality”) of that taxon is questioned. Therefore, discrepancy between universality of the taxon concept in biology and unavoidable plurality of its interpretations can be regarded as a source of problem of the taxon ontology. The present work aims to clarify the premises of this discrepancy using phenomenological approach. Three ways of the taxon positing (as a class, as a place in the world, and as a individualized body) have been distinguished. Taxon as a class is established by common essence that is shared by a set of living beings. These living beings are regarded as speculative objects beyond an idea of the world, i.e. as objects of the experimental science. A question about ontology of taxon as a class refers to the scholastic problem of universalia; its status can be defined within the scope of the nominalism/realism opposition. Taxon as a place of common appearance of the specimens is regarded in the context of the etiological relations unifying various entities into the entire world. Taxon as a place refers to a certain position in the Natural System that is construed as an etiological map of the world. In this case a specimen of a living being is known as a curiosity, i.e. representant of its relationships as well as of the place of its origin. Ontological status of a taxon as a place is to be clarified within the framework of the natural/artificial opposition. The positing of a taxon as a collective body marked off by limits of joint survival of living beings is characteristic for biology in the strict sense which arose in the very beginning of the 19 century. A taxon as a body established by the techniques of disciplinary power sensu M. Foucault extended from the human bodies to bodies of other living beings. The ontological status of a taxon as a collective body can be defined within the scope of the wild/domesticated opposition. Therefore, the discrepancy between the universality of the taxon concept and the plurality of its interpretations is underlayed by interpenetration of three distinct modi of taxon establishing. Distinguishing between these three modi can clarify sources of ontological problems concerned with the term taxon in each case when they arise.     

Tissue microarrays as a platform for proteomic investigation

Tissue microarrays have become an essential tool in translational pathology. They are used to confirm results from other experimental platforms, such as expression microarrays, as well as a primary tool to explore the expression profile of proteins by immunohistochemical analysis. Tissue microarrays are routinely used molecular epidemiology, drug development and determining the diagnostic, prognostic and predictive value of new biomarkers. By applying traditional protein based assays, as well as novel assays to the platform, tissue microarrays have gained a new utility as a proteomic tool for both basic science as well as clinical investigation. This article will explore the new approaches that are being applied to tissue microarrays to, characterize the human proteome, and new technologies that allow tissue microarrays to function as a protein array. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged     

Production of new polymeric compounds in plants

Plant metabolic engineering has recently enabled the synthesis of a range of polyhydroxyalkanoates as well as a protein-based polymer. These novel compounds can be harvested from plants as a renewable source of environmentally friendly polymers or can be used to change the physical properties of plant products, such as fibres.     

Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process

The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well.