Biosensor for rapid determination of 3-hydroxybutyrate using bi-enzyme system

A bi-enzyme-based Clark electrode was developed for the determination of 3-hydroxybutyrate. This sensor is based on the specific dehydrogenation by 3-hydroxybutyrate dehydrogenase (HBDH, E.C. 1.1.1.30) in combination with salicylate hydroxylase (SHL E.C. 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, HBDH, catalyses the specific dehydrogenation of 3-hydroxybutyrate consuming NAD(+). The products, NADH, initiate the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. SHL forces the equilibrium of dehydrogenation of 3-hydroxybutyrate by HBDH to the product side by consuming NADH. Dissolved oxygen acts as an essential material for SHL during its enzymatic reactions. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of 3-hydroxybutyrate. Interferences from different amino acids and electroactive substances were found to be minimal due to the specificity of HBDH and the application of a Teflon membrane. The sensor has a fast response (2s) and short recovery time (2 min) with a linear range between 8 and 800 microM 3-hydroxybutyrate and a detection limit of 3.9 microM. A good agreement (R(2)=0.9925) with theoretical calculation was obtained in spiked serum sample measurements.     

Effects of ethanol on lipid bilayers with and without cholesterol: the distearoylphosphatidylcholine system

Differential scanning calorimetry (DSC) and fluorescence spectroscopy are useful techniques for investigating the phase transitions of phospholipid bilayers. In this study, these methods have been extended to determine the effects of ethanol on DSPC and DSPC/2 mol.% cholesterol bilayers. The biphasic effect of the main transition was observed on the DSC heating scans above 0.60 M ethanol. In addition, the concentration at which the biphasic effect occurs is not significantly changed in the presence of 2 mol.% cholesterol. For the fluorescence studies, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into the bilayer to monitor the phase transitions through the displacement of DPH. This fluorescent probe is used to directly determine the onset of interdigitation in the bilayer systems as indicated by a large decrease in the DPH fluorescence intensity. The addition of cholesterol lowered and broadened the transition temperatures of the phosphatidylcholine (PC) system. However, 2 mol.% cholesterol did not have a significant effect on the induction of the interdigitated phase in DSPC as observed from the small difference in ethanol threshold concentration for the two systems. This suggests that DSPC forms a more stable interdigitated gel phase than other PCs with shorter acyl chains.     

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.     

Acinetobacter baylyi Starvation-Induced Genes Identified through Incubation in Long-Term Stationary Phase

Acinetobacter species encounter cycles of feast and famine in nature. We show that populations of Acinetobacter baylyi strain ADP1 remain dynamic for 6 weeks in batch culture. We created a library of lacZ reporters inserted into SalI sites in the genome and then isolated 30 genes with lacZ insertions whose expression was induced by starvation during long-term stationary phase compared with their expression during exponential growth. The genes encode metabolic, gene expression, DNA maintenance, envelope, and conserved hypothetical proteins.Acinetobacter species are ubiquitous soil organisms. Starvation during long-term stationary phase (LTSP) can serve as a laboratory model for natural competitive conditions such as those found in soils (4). This model has been used to study Escherichia coli, and here, we have applied it to Acinetobacter baylyi strain ADP1 (8).During long-term batch culture, an initially clonal population of Escherichia coli experiences five growth stages: lag, exponential, and stationary phases and then death phase and LTSP (4). Prior to LTSP, most of the cells die and serve as nutrition for starving survivors (6, 13). In LTSP, the cell population remains almost steady, declining slowly over years (reviewed in reference 4): for each newly dead cell, slightly less than one new cell is “born.”Much of what is known about starvation physiology during LTSP has been determined through study of the growth advantage in stationary phase (GASP) phenotype. The phenotype arises from genetic changes that occur when cells experience LTSP. During LTSP, the population may have a mutation frequency approaching 1 in 600 base pairs per genome (5).Some physiological changes that take place during LTSP have been described, as have some genes necessary for the development of GASP (13, reviewed in reference 12). Some mutant strains that exhibit GASP have mutations that enhance catabolic efficiency for processing amino acids (14-16). Another nutrient consumed is DNA, which requires genes homologous to strain ADP1''s competence genes (6). Additionally, mutations that knock out SOS polymerases interfere with the formation of GASP mutants (11).     

Cell mediated immune response elicited in mice after immunization with the P64k meningococcal protein: epitope mapping

The P64k protein of Neisseria meningitidis has been reported as an immunological carrier for weak immunogens. This investigation was aimed at characterizing the T-cell response produced in primed mice and at identifying T helper cell epitopes within this molecule. BALB/c mice subcutaneously immunized with the recombinant antigen provided inguinal lymph node cells (LNC) that proliferated in the presence of P64k in a dose-dependent manner. Proliferating cells secreted IL-4 while the concentration of IL-12 remained unaltered in the culture supernatant. By testing a panel of 59 overlapping synthetic peptides spanning the entire sequence of the antigen a T-cell determinant was localized. Prime-boost and lymphoproliferation experiments, conducted with highly purified synthetic peptides, confirmed that the segment including amino acids 470-485 comprises a T-cell epitope within the P64k molecule.     

Mismatch of morphological and molecular identifications in native and invasive subspecies of Codium fragile (Bryopsidophyceae,Chlorophyta)

Several subspecies are defined within Codium fragile, including the invasive C. fragile ssp. fragile, first reported in New Zealand in 1973. An endemic subspecies, C. fragile ssp. novae‐zelandiae, is also found throughout New Zealand. The two subspecies exhibit morphological and molecular variation, although these have never been evaluated together. We compared variation between subspecies at locations in Auckland, identifying subspecies using rps3‐rpl16 DNA sequence data, and assessing gross morphological differences, anatomical utricle characters and morphometrics. The taxonomic utility of the morphometric data sets was assessed by linear discriminant analysis. Utricle characters and measurements varied within individual thalli and between different preservation methods. The phenotypes of both subspecies were highly variable and influenced by environment. Accurate subspecies delimitation using morphological data was not possible; the discriminant analyses performed no better than chance for all combinations of the morphological data. Specimens from New Zealand, Canada, Australia and Ireland were sequenced using both the rps3‐rpl16 and tufA plastid markers. The tufA elongation factor was shown to be a good candidate for differentiating subspecies of C. fragile. This marker is twice the length of the rps3‐rpl16 spacer, shows greater variation between ssp. fragile and novae‐zelandiae, and is less prone to sequencing error. A simple restriction enzyme digest of the tufA amplicon can distinguish ssp. fragile and ssp. novae‐zelandiae. Our study expands the known range of the ssp. fragile in New Zealand, including the first record of this subspecies from the west coast of Auckland, and points to a need to re‐evaluate morphological and molecular criteria for subspecies currently defined within C. fragile.     

Postglacial recolonization of eastern Blacknose Dace,Rhinichthys atratulus(Teleostei: Cyprinidae), through the gateway of New England

During the last ice age, much of North America far south as 40°N was covered by glaciers (Hewitt 2000). About 20,000 years ago, as the glaciers retreated, the hydrologic landscape changed dramatically creating waterways for fish dispersal. The number of populations responsible for recolonization and the regions from which they recolonized are unknown for many freshwater fishes living in New England and southeastern Canada. The Blacknose Dace,Rhinichthys atratulus, is one of the freshwater fish species that recolonized this region. We hypothesize that the earliest deglaciated region, modern-day Connecticut, was recolonized byR. atratulusvia a single founding event by a single population. In this paper, we test this hypothesis phylogenetically with regard to the major drainage basins within Connecticut. The mitochondrial DNA exhibits low nucleotide diversity, high haplotype diversity, and a dominant haplotype found across the state. A small percentage of individuals in the Housatonic drainage basin, however, share a haplotype with populations in New York drainage basins, a haplotype not found elsewhere in Connecticut's drainage basins. We calculated a range for the rate of divergence for NADH dehydrogenase subunit 2 (nd2) and control region (ctr) of 4.43-6.76% and 3.84-8.48% per million years (my), respectively. While this range is higher than the commonly accepted rate of 2% for mitochondrial DNA, these results join a growing list of publications finding high rates of divergence for various taxa (Peterson and Masel 2009). The data support the conclusion that Connecticut as a whole was recolonized initially by a single founding event that came from a single refugium. Subsequently, the Housatonic basin alone experienced a secondary recolonization event.     

Effects of Landscape Fragmentation and Climate on Lyme Disease Incidence in the Northeastern United States

Lyme disease is the most frequently reported vector borne illness in the United States, and incidences are increasing steadily year after year. This study explores the influence of landscape (e.g., land use pattern and landscape fragmentation) and climatic factors (e.g., temperature and precipitation) at a regional scale on Lyme disease incidence. The study area includes thirteen states in the Northeastern United States. Lyme disease incidence at county level for the period of 2002–2006 was linked with several key landscape and climatic variables in a negative binomial regression model. Results show that Lyme disease incidence has a relatively clear connection with regional landscape fragmentation and temperature. For example, more fragmentation between forests and residential areas results in higher local Lyme disease incidence. This study also indicates that, for the same landscape, some landscape variables derived at a particular scale show a clearer connection to Lyme disease than do others. In general, the study sheds more light on connections between Lyme disease incidence and climate and landscape patterns at the regional scale. Integrating findings of this regional study with studies at a local scale will further refine understanding of the pattern of Lyme disease as well as increase our ability to predict, prevent, and respond to disease.     

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH₂), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.     

Interval-based distance function for identifying RNA structure candidates

Many clustering approaches have been developed for biological data analysis, however, the application of traditional clustering algorithms for RNA structure data analysis is still a challenging issue. This arises from the existence of complex secondary structures while clustering. One of the most critical issues of cluster analysis is the development of appropriate distance measures in high dimensional space. The traditional distance measures focus on scale issues, but ignores the correlation between two values. This article develops a novel interval-based distance (Hausdorff) measure for computing the similarity between characterized structures. Three relationships including perfect match, partially overlapped and non-overlapped are considered. Finally, we demonstrate the methods by analyzing a data set of RNA secondary structures from the Rfam database.