Mechanical collapse of confined fluid membrane vesicles

Compact cylindrical and spherical invaginations are common structural motifs found in cellular and developmental biology. To understand the basic physical mechanisms that produce and maintain such structures, we present here a simple model of vesicles in confinement, in which mechanical equilibrium configurations are computed by energy minimization, balancing the effects of curvature elasticity, contact of the membrane with itself and the confining geometry, and adhesion. For cylindrical confinement, the shape equations are solved both analytically and numerically by finite element analysis. For spherical confinement, axisymmetric configurations are obtained numerically. We find that the geometry of invaginations is controlled by a dimensionless ratio of the adhesion strength to the bending energy of an equal area spherical vesicle. Larger adhesion produces more concentrated curvatures, which are mainly localized to the “neck” region where the invagination breaks away from its confining container. Under spherical confinement, axisymmetric invaginations are approximately spherical. For extreme confinement, multiple invaginations may form, bifurcating along multiple equilibrium branches. The results of the model are useful for understanding the physical mechanisms controlling the structure of lipid membranes of cells and their organelles, and developing tissue membranes.     

COMPARISON OF TWO SIMPLE METHODS FOR THE MEASUREMENT OF DETECTION THRESHOLDS FOR BASIC, UMAMI AND METALLIC TASTES

Two simple methods were followed to determine detection thresholds for the taste of substances in aqueous solution. The methods applied were: a modification of the ascending method of limits and a method based on the use of scales. Detection thresholds were calculated for the four basic tastes (sweet, salty, acid, and bitterness), umami and metallic. Reference substances for each taste were sucrose, sodium chloride, citric acid, caffeine, monosodium glutamate and iron (II) sulfate heptahydrate and the results of the two methods were compared. We found that the threshold values calculated by method ASTM-679 was within the range of concentrations identified with the scales method.     

Nucleotide sequence reveals overlap between T4 phage genes encoding dihydrofolate reductase and thymidylate synthase

We have determined the nucleotide sequence of a 1075-base-pair HindIII fragment of the T4 phage genome. This fragment contains the structural gene (frd) for dihydrofolate reductase and part of the gene (td) encoding thymidylate synthase. The fragment contains a 579-base-pair open reading frame, encoding a 193-residue polypeptide with a calculated mass of 21,603 Da, in agreement with our reported subunit molecular mass of 23,000. The deduced amino acid sequence shows partial homology with other dihydrofolate reductases, with most of the identities lying in regions known to be involved in substrate binding and catalysis. The 3' end of the coding strand overlaps the coding region for thymidylate synthase; the sequence - ATGA -includes an opal terminator for the frd gene and an initiating triplet for the td gene. The deduced amino acid sequence from this initiating ATG is identical, for the first 20 residues, with the NH2-terminal 20 residues reported for the td protein (M. Belfort , A. Moelleken , G. F. Maley , and F. Maley (1983) J. Biol. Chem. 258, 2045-2051). The sequenced HindIII fragment was transferred into a high expression plasmid vector for large scale production of homogeneous T4 dihydrofolate reductase. The experimentally determined sequence of 20 residues at the NH2-terminus of this protein is identical with that deduced from the nucleotide sequence for T4 dihydrofolate reductase.     

Activation-dependent changes in microenvironment of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase

The spinach ribulose 1,5-bisphosphate carboxylase/oxygenase was labelled with o-phthalaldehyde, which forms a stable fluorescent isoindole adduct at the active site. The fluorescence behaviour of the labelled enzyme after activation to different levels by Mg2+ was compared with that of a synthetic isoindole adduct of o-phthalaldehyde, namely 1-(hydroxyethylthio)-2-beta hydroxyethylisoindole in solvents of different pH and polarity. The results suggest that the microenvironment at the catalytically incompetent active site of the unactivated Rubisco is highly acidic (pH less than 2) in nature. The activation by Mg2+ results in the conformational change such that the effective pH at the active site increases to greater than 8. The polarity of the active site of the activated enzyme was found to be similar to that of a mixture of hexane and toluene.     

Distinguishing features of 16S rDNA gene for five dominating bacterial genus observed in bioremediation.

Defining a microbial community and identifying bacteria, at least at the genus level, is a first step in predicting the behavior of a microbial community in bioremediation. In biological treatment systems, the most dominating groups observed are Pseudomonas, Moraxella, Acinetobactor, Burkholderia, and Alcaligenes. Our interest lies in identifying the distinguishing features of these bacterial groups based on their 16S rDNA sequence data, which could be used further for generating genus-specific probes. Accordingly, 20 sequences representing different species from each genus above were retrieved, which constituted a training set. A 16-dimensional feature vector comprised of transition probabilities of nucleotides was considered and each sampled sequence was expressed in terms of these features. A stepwise feature selection method was used to identify features that are distinct across the species of these five groups. Wilk's lambda selection criterion was used and resulted in a subset with six distinguishing features. The discriminating efficacy of this subset was tested through multiple group discriminant analysis. Two linear composites, as a function of these features, could discriminate the test set of forty-five sequences from these groups with 95% accuracy, thereby ascertaining the relevance of the identified features. The geometric representation of feature correlation in the reduced discriminant space demonstrated the dominance of identified features in specific groups. These features independently or in combination could be used to generate genus-specific patterns to design probes, so as to develop a tracking tool for the selected group of bacteria.     

Heterogeneity of large subunits of ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha

Homogeneous ribulose-1,5-bisphosphate carboxylase purified from autotrophically grown $\text{Hydrogenomonas eutropha}$ can be dissociated with sodium dodecylsulfate into small 15,000-dalton subunits and large 56,000- and 52,000-dalton subunits (the latter in a mole ratio of 5:3). The overal mole ratio of small to large subunits is 1.08. Considering the molecular weight of the native enzyme (516,000), the simplest quarternary structure of this enzyme consists of 8 large (mixed) and 8 small subunits. Isolation of the enzyme from cells under conditions that should minimize proteolysis has no effect upon the observed heterogeneity of the large subunits.     

Ribulose bisphosphate carboxylase/oxygenase from Thiocapsa roseopersicina

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified 80-fold from malate-grown Thiocapsa roseopersicina by salting out the enzyme from the high-speed supernatant between 68–95% saturation with respect to (NH₄)₂SO₄, gelfiltration through Sephadex G-100, and DEAE-cellulose chromatography followed by sedimentation into a 14–34% glycerol gradient. The specific activity of enzyme for the carboxylase reaction was 2.45 mol RuBP-dependent CO₂ fixed/min · mg protein (at pH 8.0 and 30° C) and for the oxygenase reaction was 0.23 mol RuBP-dependent O₂ consumed/min · mg protein (at pH 8.6, and 25° C). The enzyme, which was ultracentrifugally homogeneous in the presence of 4 and 10% v/v glycerol, was stable for at least one year at-80° C in the presence of 10% glycerol. S_{20, w} values obtained in the presence of 4 and 10% glycerol were 19.3 and 16.2, respectively. The enzyme contained both large (53,000-daltons) and mixed small subunits (15,000- and 13,500-daltons).Borate-dependent inactivation of the enzyme by 2,3-butadione, which was greatly reduced in the presence of the product 3-phosphoglycerate, suggested that one or more arginines are at the active site.Abbreviations DTT dithiotreitol - RuBP d-ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate - TCA trichloroacetic acid - TEMBDG buffer (pH 8.0 at 25°C) containing 20 mM Tris, 1 mM disodium EDTA · 2 H₂O, 10 mM MgCl₂·6 H₂O, 50 mM NaHCO₃, 0.1 mM DTT and 10% glycerol (v/v)     

Strain-specific differences in <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> associated with the phase variable gene repertoire

Background  

There are several differences associated with the behaviour of the four main experimental Neisseria gonorrhoeae strains, FA1090, FA19, MS11, and F62. Although there is data concerning the gene complements of these strains, the reasons for the behavioural differences are currently unknown. Phase variation is a mechanism that occurs commonly within the Neisseria spp. and leads to switching of genes ON and OFF. This mechanism may provide a means for strains to express different combinations of genes, and differences in the strain-specific repertoire of phase variable genes may underlie the strain differences.     

Genetic islands of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> strains NEM316 and 2603VR and their presence in other Group B Streptococcal strains

Background  

Streptococcus agalactiae (Group B Streptococcus; GBS) is a major contributor to obstetric and neonatal bacterial sepsis. Serotype III strains cause the majority of late-onset sepsis and meningitis in babies, and thus appear to have an enhanced invasive capacity compared with the other serotypes that cause disease predominantly in immunocompromised pregnant women. We compared the serotype III and V whole genome sequences, strains NEM316 and 2603VR respectively, in an attempt to identify genetic attributes of strain NEM316 that might explain the propensity of strain NEM316 to cause late-onset disease in babies. Fourteen putative pathogenicity islands were described in the strain NEM316 whole genome sequence. Using PCR- and targeted microarray- strategies, the presence of these islands were assessed in a diverse strain collection including 18 colonizing isolates from healthy pregnant women, and 13 and 8 invasive isolates from infants with early- and late-onset sepsis, respectively.