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.     

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.     

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.     

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.     

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.     

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     

The structure and function of tRNA-like domain in E. coli tmRNA.

tmRNA has a dual function both as a tRNA and as an mRNA in trans-translation. tmRNA possesses structural elements similar to canonical tRNAs. Our mutation studies show that the tRNA-like structure is crucial for function as an mRNA as well as a tRNA.     

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.     

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.     

A Deep-Structured Conditional Random Field Model for Object Silhouette Tracking

In this work, we introduce a deep-structured conditional random field (DS-CRF) model for the purpose of state-based object silhouette tracking. The proposed DS-CRF model consists of a series of state layers, where each state layer spatially characterizes the object silhouette at a particular point in time. The interactions between adjacent state layers are established by inter-layer connectivity dynamically determined based on inter-frame optical flow. By incorporate both spatial and temporal context in a dynamic fashion within such a deep-structured probabilistic graphical model, the proposed DS-CRF model allows us to develop a framework that can accurately and efficiently track object silhouettes that can change greatly over time, as well as under different situations such as occlusion and multiple targets within the scene. Experiment results using video surveillance datasets containing different scenarios such as occlusion and multiple targets showed that the proposed DS-CRF approach provides strong object silhouette tracking performance when compared to baseline methods such as mean-shift tracking, as well as state-of-the-art methods such as context tracking and boosted particle filtering.     

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.     

FSI analysis of a human trachea before and after prosthesis implantation

In this work we analyzed the response of a stenotic trachea after a stent implantation. An endotracheal stent is the common treatment for tracheal diseases such as stenosis, chronic cough, or dispnoea episodes. Medical treatment and surgical techniques are still challenging due to the difficulties in overcoming potential complications after prosthesis implantation. A finite element model of a diseased and stented trachea was developed starting from a patient specific computerized tomography (CT) scan. The tracheal wall was modeled as a fiber reinforced hyperelastic material in which we modeled the anisotropy due to the orientation of the collagen fibers. Deformations of the tracheal cartilage rings and of the muscular membrane, as well as the maximum principal stresses, are analyzed using a fluid solid interaction (FSI) approach. For this reason, as boundary conditions, impedance-based pressure waveforms were computed modeling the nonreconstructed vessels as a binary fractal network. The results showed that the presence of the stent prevents tracheal muscle deflections and indicated a local recirculatory flow on the stent top surface which may play a role in the process of mucous accumulation. The present work gives new insight into clinical procedures, predicting their mechanical consequences. This tool could be used in the future as preoperative planning software to help the thoracic surgeons in deciding the optimal prosthesis type as well as its size and positioning.     

A note on visual integrative mechanisms

In a system as complex and as effective as the eye, the cooperative interaction of different mechanisms may be taken as axiomatic. With this as a starting point, various visual phenomena are considered, such as short term memory, eye movements, and flicker fusion. Simple data on mean values lend support to the proposition that the spatial and temporal characteristics of these phenomena are matched with one another. The significance of this for a mechanism of vision is discussed.     

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.     

Utilization of organophosphonates by environmental micro-organisms

A survey of the utilization by environmental micro-organisms of a range of compounds containing the carbon–phosphorus (C–P) bond was carried out. Elective culture studies indicated that 15 of 19 alkylphosphonates tested served only as a sole source of phosphorus for microbial growth. Their metabolism did not lead to the extracellular release of inorganic phosphate. However, four organophosphonates—phosphonoacetate, phosphonoalanine, 2-aminoethylphosphonate and phosphonomycin—supported microbial growth when supplied as either a phosphorus source or as a carbon and energy source, with near-quantitative inorganic phosphate release. Four of five aminoalkylphosphonates tested were also utilized as a nitrogen source in the presence of 1 mmol l⁻¹ inorganic phosphate. In a subsequent screening programme, 99% of bacterial isolates tested were able to utilize 2-aminoethylphosphonate as a sole phosphorus source, 61% as a nitrogen source, 10% as a source of nitrogen and phosphorus, and 2% as a source of carbon, nitrogen and phosphorus ; 2% of isolates used phosphonoalanine as a nitrogen source. These results suggest that the uptake and metabolism of organophosphonates by bacteria is less `tightly' regulated by phosphorus starvation than has previously been supposed.     

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.     

Cleaning behaviour of the cichlid Mesonauta festivus in the Pantanal wetlands: evidence of a potential freshwater cleaning station

Cleaning interactions are known among several groups of fishes, with a higher number of records for marine fish species. These temporary associations occur between one species that acts as the cleaner and the other species as the client. The interaction usually takes place within the boundaries defined by the cleaner. This site, known as the cleaning station, allows the client fish to strike a typical pose allowing the cleaner to approach, such as wide-open fins and inclined, motionless body. Few studies have reported on the cleaning behaviour of freshwater fish species, and none has reported on behaviour that could be interpreted as establishing a cleaning station. Herein we present a new record of the cleaning interaction in the Neotropics, between the cichlid Mesonauta festivus as the cleaner and three species of anostomid (Leporinus macrocephalus, Leporinus friderici and Schizodon borellii) as clients on Pantanal wetlands, as well as the establishment of a defined cleaning station.