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.     

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     

Counselling the parents of handicapped children.

The parents of handicapped children have to adjust to a wide variety of emotional and psychological problems when first confronted with the failure of their reproductive expectations. Counselling is a formal procedure or transaction in which both counsellor and parents aim to find a mutually acceptable plan of adjustment. Parents may need support to cope with their own personal inadequacies as well as with their own personal inadequacies as well as with the needs of the child. Counselling should be a continuous process, in which the parents can learn to accept the child as a different rather than a lesser person.     

Virtues and limitations of the preimplantation mouse embryo as a model system

The mouse is the most widely used model of preimplantation embryo development, but is it a good model? Its small size, prolificacy and ease of handling make the mouse a relatively low cost, readily available and attractive alternative when embryos from other species are difficult or expensive to obtain. However, the real power of the mouse as a model lies in mouse genetics. The development of inbred mouse strains facilitated gene discovery as well as our understanding of gene function and regulation while the development of tools to introduce precise genetic modifications uniquely positioned the mouse as a powerful model system for uncovering gene function. However, all models have limitations; the small size of the mouse limits tissue availability and manipulations that can be performed and differences in physiology among species may make it inappropriate to extrapolate from the mouse to other species. Thus, rather than extrapolating directly from the mouse to other species, it may be more useful to use the mouse as a model system for developing and refining hypotheses to be tested directly in species of interest. In this brief review, the value of the preimplantation mouse embryo as a model is considered, both as a model for other species and as a model for the mouse, as understanding the virtues and limitations of the mouse as a model system is essential to its appropriate use.     

t-mixing: from fluctuation relations to response and irreversibility in MD

This paper provides an introduction to the theory of steady-state fluctuation relations for molecular dynamics systems, that led to a general theory of response. The main ingredient of this theory is a new dynamical condition now known as t-mixing. We use such a condition to identify necessary and sufficient conditions for the relaxation to a steady state (whether equilibrium or not) of an ensemble of identical systems, as well as of a single system. This allows us to address the problem of the irreversibility of time reversal invariant (conservative as well as dissipative) particle systems.     

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.     

Regulation of caspase activation in apoptosis: implications for transformation and drug resistance

Recent developments in the apoptosis field have uncovered a family of cysteine proteases, the Caspases, that act as signalling components as well as effectors of the cell death machinery. Caspases are constitutively present as inactive precursors within most cells and undergo proteolytic processing in response to diverse death-inducing stimuli to initiate the death programme. Active caspases can process other caspases of the same type as well as process caspases further downstream in the pathway that ultimately leads to collapse of the cell. This cellular collapse is thought to occur as a consequence of caspase-mediated cleavage of a diverse array of cellular substrates. Regulation of entry into the death programme is controlled at a number of levels by members of the Bcl-2 family, as well as by other cell death regulatory proteins. Recent data has shed light upon the mechanism of action of these regulatory molecules and suggests that the point of caspase activation is a major checkpoint in the cell death programme. Because many transformed cell populations possess derangements in cell death-regulatory genes, such as bcl-2, such cells frequently exhibit elevated resistance to cytotoxic chemotherapy. Thus, a deeper understanding of how apoptosis is normally regulated has therapeutic implications for disease states where the normal controls on the cell death machinery have been subverted. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Cytokinesis in prokaryotes and eukaryotes: common principles and different solutions.

Cytokinesis requires duplication of cellular structures followed by bipolarization of the predivisional cell. As a common principle, this applies to prokaryotes as well as eukaryotes. With respect to eukaryotes, the discussion has focused mainly on Saccharomyces cerevisiae and on Schizosaccharomyces pombe. Escherichia coli and to a lesser extent Bacillus subtilis have been used as prokaryotic examples. To establish a bipolar cell, duplication of a eukaryotic origin of DNA replication as well as its genome is not sufficient. Duplication of the microtubule-organizing center is required as a prelude to mitosis, and it is here that the dynamic cytoskeleton with all its associated proteins comes to the fore. In prokaryotes, a cytoskeleton that pervades the cytoplasm appears to be absent. DNA replication and the concomitant DNA segregation seem to occur without help from extensive cytosolic supramacromolecular assemblies but with help from the elongating cellular envelope. Prokaryotic cytokinesis proceeds through a contracting ring, which has a roughly 100-fold-smaller circumference than its eukaryotic counterpart. Although the ring contains proteins that can be considered as predecessors of actin, tubulin, and microtubule-associated proteins, its macromolecular composition is essentially different.     

Electrochemical reduction of xylose to xylitol by whole cells or crude enzyme of Candida peltata

In this study, whole cells and a crude enzyme of Candida peltata were applied to an electrochemical bioreactor, in order to induce an increment of the reduction of xylose to xylitol. Neutral red was utilized as an electron mediator in the whole cell reactor, and a graphite-Mn(IV) electrode was used as a catalyst in the enzyme reactor in order to induce the electrochemical reduction of NAD(+) to NADH. The efficiency with which xylose was converted to xylitol in the electrochemical bioreactor was five times higher than that in the conventional bioreactor, when whole cells were employed as a biocatalyst. Meanwhile, the xylose to xylitol reduction efficiency in the enzyme reactor using the graphite-Mn (IV) electrode and NAD(+) was twice as high as that observed in the conventional bioreactor which utilized NADH as a reducing power. In order to use the graphite-Mn(IV) electrode as a catalyst for the reduction of NAD(+) to NADH, a bioelectrocatalyst was engineered, namely, oxidoreductase (e.g. xylose reductase). NAD(+) can function in this biotransformation procedure without any electron mediator or a second oxidoreductase for NAD(+)/NADH recycling.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

ONTOLOGY OR PHENOMENOLOGY? HOW THE LVAD CHALLENGES THE EUTHANASIA DEBATE

This article deals with the euthanasia debate in light of new life‐sustaining technologies such as the left ventricular assist device (LVAD). The question arises: does the switching off of a LVAD by a doctor upon the request of a patient amount to active or passive euthanasia, i.e. to ‘killing’ or to ‘letting die’? The answer hinges on whether the device is to be regarded as a proper part of the patient's body or as something external. We usually regard the switching off of an internal device as killing, whereas the deactivation of an external device is seen as ‘letting die’. The case is notoriously difficult to decide for hybrid devices such as LVADs, which are partly inside and partly outside the patient's body. Additionally, on a methodological level, I will argue that the ‘ontological’ arguments from analogy given for both sides are problematic. Given the impasse facing the ontological arguments, complementary phenomenological arguments deserve closer inspection. In particular, we should consider whether phenomenologically the LVAD is perceived as a body part or as an external device. I will support the thesis that the deactivation of a LVAD is to be regarded as passive euthanasia if the device is not perceived by the patient as a part of the body proper.     

Isolation and characterization of Deinococcus geothermalis T27, a slightly thermophilic and organic solvent-tolerant bacterium able to survive in the presence of high concentrations of ethyl acetate

A solvent-tolerant, slightly thermophilic bacterium was isolated at 45 degrees C in the presence of toluene vapor provided as the sole carbon source. Strain T27 was identified as Deinococcus geothermalis T27. It could tolerate high concentrations of solvent provided as a nonaqueous layer (5% and 20%, v/v) to a cell suspension and had a remarkable ability to tolerate a broad range of solvents having log P(ow) values ranging from 5.6 of n-decane to as low as 0.7 of ethyl acetate. It was also able to utilize some of the solvents tested as a growth substrate at 45 degrees C. The addition of Ca(2+) ion, glucose and fructose partially promoted solvent tolerance. Cells exposed to ethyl acetate appeared to have a smaller size; however, the cell structure was not altered and was apparently well defined even after solvent shock. The tolerance of D. geothermalis T27 in the presence of high levels of toxic solvent stress at a comparatively high temperature indicated its potential use in biotechnological applications as well as bioremediation of xenobiotics.     

The Acoustic Codes: How Animal Sign Processes Create Sound-Topes and Consortia via Conflict Avoidance

In this essay we argue for the possibility to describe the co-presence of species in a community as a consortium built by acoustic codes, using mainly the examples of bird choruses. In this particular case, the consortium is maintained via the sound-tope that different bird species create by singing in a chorus. More generally, the formation of acoustic codes as well as cohesive communicative systems (the consortia) can be seen as a result of plastic adaptational behaviour of the specimen who can solve and avoid conflicts both with conspecifics and with other species in the vicinity. Thus, sign-relations appear to resolve potential conflicts, and as a foundation for symbiotic aggregations. The spatio-temporal configuration of consortia—their chronotope—includes several eco-fields as respective to different functions of the participating organisms. Biological study is combined with a semiotic approach that, as we suggest, should be more often used together to effectively describe ecological processes.     

A possible involvement of glutathione in the detoxication of sulfite

Inorganic sulfite may be detoxified by conversion to S-sulfocysteine. We demonstrate this conversion by a series of enzyme-catalyzed steps as follows. Inorganic sulfite reacts with glutathione disulfide by a thiol transferase catalyzed reaction as previously demonstrated. The S-sulfoglutathione formed is then converted by gamma-glutamyltranspeptidase to S-sulfocysteinylglycine and the latter finally hydrolyzed to S-sulfocysteine by a renal dipeptidase. S-Sulfoglutathione is a substrate for gamma-glutamyl-transpeptidase as effective as glutathione itself. Furthermore, S-sulfocysteinylglycine is cleaved as efficiently as cysteinylglycine by a renal dipeptidase at high substrate concentrations but somewhat less efficiently at low substrate concentrations.     

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.     

Phylogeny of the Celastraceae inferred from phytochrome B gene sequence and morphology

Phylogenetic relationships within Celastraceae were inferred using a simultaneous analysis of 61 morphological characters and 1123 base pairs of phytochrome B exon 1 from the nuclear genome. No gaps were inferred, and the gene tree topology suggests that the primers were specific to a single locus that did not duplicate among the lineages sampled. This region of phytochrome B was most useful for examining relationships among closely related genera. Fifty-one species from 38 genera of Celastraceae were sampled. The Celastraceae sensu lato (including Hippocrateaceae) were resolved as a monophyletic group. Loesener's subfamilies and tribes of Celastraceae were not supported. The Hippocrateaceae were resolved as a monophyletic group nested within a paraphyletic Celastraceae sensu stricto. Goupia was resolved as more closely related to Euphorbiaceae, Corynocarpaceae, and Linaceae than to Celastraceae. Plagiopteron (Flacourtiaceae) was resolved as the sister group of Hippocrateoideae. Brexia (Brexiaceae) was resolved as closely related to Elaeodendron and Pleurostylia. Canotia was resolved as the sister group of Acanthothamnus within Celastraceae. Perrottetia and Mortonia were resolved as the sister group of the rest of the Celastraceae. Siphonodon was resolved as a derived member of Celastraceae. Maytenus was resolved as three disparate groups, suggesting that this large genus needs to be recircumscribed.