A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum

The detection of a little as 0.2 pg (60,000 molecules) of hepatitis B viral (HBV) DNA in human serum samples in 4 h has been demonstrated using a solution-hybridization and bead-capture method. An amplification method based on chemically crosslinked oligodeoxyribonucleotides was coupled with a horseradish peroxidase-labeling scheme for the ultimate detection of the analyte. Two sets of HBV complementary synthetic oligodeoxyribonucleotide probes containing one of two types of single-stranded (ss) overhangs were employed. These ss overhangs were used to capture the probe-analyte complex onto a bead and subsequently to label it. Detection was achieved with either a chemiluminescent or colorimetric output substrate for the enzyme. Only in the presence of the virus was label specifically bound to the support. The assay was relatively unaffected by either sample composition or by the presence of heterologous nucleic acids.     

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.     

Edible Lepidoptera in Mexico: Geographic distribution, ethnicity, economic and nutritional importance for rural people

In this paper, we reported the butterflies and moths that are consumed in Mexico. We identified 67 species of Lepidoptera that are eaten principally in their larval stage in 17 states of Mexico. These species belong to 16 families: Arctiidae, Bombycidae, Castniidae, Cossidae, Geometridae, Hepialidae, Hesperiidae, Lasiocampidae, Noctuidae, Nymphalidae, Papilionidae, Pieridae, Pyralidae, Saturniidae, Sesiidae, and Sphingidae. Saturniidae, Pieridae, Noctuidae and Nymphalidae were the more species consumed with 16, 11, 9, and 8 species, respectively. The genera with the largest numbers of species were: Phassus, Phoebis, Hylesia and Spodoptera, with three species. Their local distribution, corresponding to each state of Mexico, is also presented.     

Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and <Emphasis Type="Italic">in vivo</Emphasis> visualization by magnetic resonance imaging

Background  

Stem cell therapy has emerged as a promising addition to traditional treatments for a number of diseases. However, harnessing the therapeutic potential of stem cells requires an understanding of their fate in vivo. Non-invasive cell tracking can provide knowledge about mechanisms responsible for functional improvement of host tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used to label and visualize various cell types with magnetic resonance imaging (MRI). In this study we performed experiments designed to investigate the biological properties, including proliferation, viability and differentiation capacity of mesenchymal cells (MSCs) labeled with clinically approved SPIONs.     

Fat taste in humans: Sources of within- and between-subject variability

Non-esterified fatty acids (NEFA) are reportedly detectable through taste mechanisms in the human oral cavity. However, wide variability has been observed in NEFA taste sensitivity between and within subjects as well as across research groups. Some of this variability may be due to the hydrophobic nature of the NEFA and the methods used to make stimuli emulsions. As NEFA are poorly soluble in water, emulsification is necessary for delivery of stimuli to taste receptors. However, properties of emulsions may also be detected by somatosensory cues complicating attribution of sensory findings to taste. Additionally, learning (improved test performance) has been observed when using traditional tests for measuring sensitivity to NEFA, which may contribute greatly to within-subject variability if not standardized. Factors such as sex, diet, and BMI have been proposed to affect NEFA taste sensitivity, but the degree to which these individual factors influence NEFA detection thresholds remains to be fully established. Improved knowledge of stimulus properties and individual sensory capabilities will be needed to further evaluate the posited taste component to human oral fat detection. Progress in this area should facilitate the translation of findings on how NEFA taste may contribute to or reflect food choice and chronic disease risk.     

Dual lipid modification of Arabidopsis Ggamma-subunits is required for efficient plasma membrane targeting

Posttranslational lipid modifications are important for proper localization of many proteins in eukaryotic cells. However, the functional interrelationships between lipid modification processes in plants remain unclear. Here we demonstrate that the two heterotrimeric G-protein gamma-subunits from Arabidopsis (Arabidopsis thaliana), AGG1 and AGG2, are prenylated, and AGG2 is S-acylated. In wild type, enhanced yellow fluorescent protein-fused AGG1 and AGG2 are associated with plasma membranes, with AGG1 associated with internal membranes as well. Both can be prenylated by either protein geranylgeranyltransferase I (PGGT-I) or protein farnesyltransferase (PFT). Their membrane localization is intact in mutants lacking PFT activity and largely intact in mutants lacking PGGT-I activity but is disrupted in mutants lacking both PFT and PGGT-I activity. Unlike in mammals, Arabidopsis Ggammas do not rely on functional Galpha for membrane targeting. Mutation of the sixth to last cysteine, the putative S-acylation acceptor site, causes a dramatic change in AGG2 but not AGG1 localization pattern, suggesting S-acylation serves as an important additional signal for AGG2 to be targeted to the plasma membrane. Domain-swapping experiments suggest that a short charged sequence at the AGG2 C terminus contributes to AGG2's efficient membrane targeting compared to AGG1. Our data show the large degree to which PFT and PGGT-I can compensate for each other in plants and suggest that differential lipid modification plays an important regulatory role in plant protein localization.     

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes

N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.     

Sublethal damage during cryopreservation of rainbow trout sperm

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at -20 degreesC/min in a programmable cooler and thawed in a 25 degreesC water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. Copyright 1998 Academic Press.     

Modeled responses of terrestrial ecosystems to elevated atmospheric CO2: a comparison of simulations by the biogeochemistry models of the Vegetation/Ecosystem Modeling and Analysis Project (VEMAP)

Although there is a great deal of information concerning responses to increases in atmospheric CO₂ at the tissue and plant levels, there are substantially fewer studies that have investigated ecosystem-level responses in the context of integrated carbon, water, and nutrient cycles. Because our understanding of ecosystem responses to elevated CO₂ is incomplete, modeling is a tool that can be used to investigate the role of plant and soil interactions in the response of terrestrial ecosystems to elevated CO₂. In this study, we analyze the responses of net primary production (NPP) to doubled CO₂ from 355 to 710 ppmv among three biogeochemistry models in the Vegetation/Ecosystem Modeling and Analysis Project (VEMAP): BIOME-BGC (BioGeochemical Cycles), Century, and the Terrestrial Ecosystem Model (TEM). For the conterminous United States, doubled atmospheric CO₂ causes NPP to increase by 5% in Century, 8% in TEM, and 11% in BIOME-BGC. Multiple regression analyses between the NPP response to doubled CO₂ and the mean annual temperature and annual precipitation of biomes or grid cells indicate that there are negative relationships between precipitation and the response of NPP to doubled CO₂ for all three models. In contrast, there are different relationships between temperature and the response of NPP to doubled CO₂ for the three models: there is a negative relationship in the responses of BIOME-BGC, no relationship in the responses of Century, and a positive relationship in the responses of TEM. In BIOME-BGC, the NPP response to doubled CO₂ is controlled by the change in transpiration associated with reduced leaf conductance to water vapor. This change affects soil water, then leaf area development and, finally, NPP. In Century, the response of NPP to doubled CO₂ is controlled by changes in decomposition rates associated with increased soil moisture that results from reduced evapotranspiration. This change affects nitrogen availability for plants, which influences NPP. In TEM, the NPP response to doubled CO₂ is controlled by increased carboxylation which is modified by canopy conductance and the degree to which nitrogen constraints cause down-regulation of photosynthesis. The implementation of these different mechanisms has consequences for the spatial pattern of NPP responses, and represents, in part, conceptual uncertainty about controls over NPP responses. Progress in reducing these uncertainties requires research focused at the ecosystem level to understand how interactions between the carbon, nitrogen, and water cycles influence the response of NPP to elevated atmospheric CO₂. Received: 13 December 1996 / Accepted: 20 November 1997