Alternansucrase mutants of Leuconostoc mesenteroides strain NRRL B-21138

Alternan is a unique α-D-glucan of potential commercial interest, produced by rare strains of Leuconostoc mesenteroides. Natural isolates that produce alternan, such as NRRL B-1355, also produce dextran as a troublesome contaminant. We previously isolated mutants of strain NRRL B-1355 that are deficient in dextran production, including the highly stable strain NRRL B-21138. In the current work, we mutagenized strain NRRL B-21138 and screened survivors for further alterations in production of alternansucrase, the enzyme that catalyzes the synthesis of alternan from sucrose. Second generation mutants included highly stable strain NRRL B-21297, which produced four-fold elevated levels of alternansucrase without an increase in the proportion of dextransucrase activity. Such alternansucrase overproducing strains will facilitate studies of this enzyme, and may become valuable for the enzymatic production of alternan. Another highly stable mutant strain, NRRL B-21414, grew slowly on sucrose with negligible production of glucan or extracellular glucansucrase activity. This strain may prove useful as an expression host for glucansucrase genes. Received 30 July 1996/ Accepted in revised form 15 December 1996     

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.     

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease V_max and K_m for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in V_max is likely due to excluded volume effects, which are partially counteracted by viscosity hindering release of the NAD⁺ product. Macromolecular crowding is further complicated by the presence of a depletion layer in solutions of dextran larger than YADH, which diminishes the hindrance from viscosity. The disparate effects from 25 g/L dextran or glucose compared to 25 g/L Ficoll or sucrose reveals that soft interactions must also be considered. Data from binary mixtures of glucose, dextran, and Ficoll support this “tuning” of opposing factors. While macromolecular crowding was originally proposed to influence proteins mainly through excluded volume effects, this work compliments the growing body of evidence revealing that other factors, such as preferential hydration, chemical interactions, and the presence of a depletion layer also contribute to the overall effect of crowding.     

Chitosan-dextran Sulfate Nanoparticles for Delivery of an Anti-angiogenesis Peptide

Summary A novel nanoparticle delivery system has been developed by employing the oppositely charged polymers chitosan (CS) and dextran sulfate (DS), and a simple coacervation process. Under the conditions investigated, the weight ratio of the two polymers is identified as a determining factor controlling particle size, surface charge, entrapment efficiency and release characteristics of the nanoparticles produced. Particles of 223 nm mean diameter were produced under optimal conditions with a zeta potential of approximately −32.6 mV. A maximum of 75% anti-angiogenesis peptide entrapment efficiency was achieved with a CS:DS weight ratio of 0.59∶1. The same nanoparticle formulation also showed slow and sustained peptide release over a period of 6 days. In contrast, the formulation containing a lower ratio of CS:DS (0.5∶1) was found to have reduced entrapment efficiency and more rapid peptide release characteristics. The results of this study suggest that physicochemical and release characteristics of the CS-DS nanoparticles can be modulated by changing ratios of two ionic polymers. The novel CS-DS nanoparticles prepared by the coacervation process have potential as a carrier for small peptides.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

The physical basis of reflective communication between fish,with special reference to the horse mackerel,Trachurus trachurus

Some properties of reflecting structures in the external surfaces of Trachurus trachurus and some other fish are described. These are related to the hypothesis that such structures are useful, especially to schooling fish, for communicating information on relative positions, orientations, and movements between neighbours. In addition to the silvery layers on the main body surfaces, there are: (a) highly silvered patches on the tail, the pectoral fins and the jaws which, in the sea, will become much brighter or darker with any movement such as a tailbeat or mouth opening which changes their orientations in the ambient lightfield, and (b) structures such as the dorsal lateral line which, in the sea, will only appear bright from certain directions. To us, the colours of the ventral flanks change from bright red to blue with direction orientations and have special reflectivity curves close to those predicted by A.F. Huxley for interference reflectors which are ''ideal'' λ/4 stacks of guanine crystals and cytoplasm. The wavebands best reflected by such platelets move to shorter wavelengths with increasing angle of incidence, also in accord with these equations. At normal incidence, the outer layer of platelets reflects maximally for far-red light which penetrates only a short distance in the sea. Such layers can, however, be useful at oblique angles where they reflect maximally in the yellow and blue. The inner layer of reflectors reflects very strongly in the blue at normal incidence, but reflects in the ultra-violet at oblique angles. Some theoretical studies are made on the ways in which the patterns of reflectivity by single and superposed layers of λ/4 stacks could signal a fish''s movements or its position relative to its neighbours.     

Interaction of alpha-sarcin and gelonin with Cibacron blue

Alpha-sarcin and gelonin, two proteins which inactivate the 60S ribosomal subunit, interact with Cibacron blue and bind to blue dextran-Sepharose, from which they are partially desorbed by nucleoside triphosphates and, more efficiently, by homopolynucleotides. It is further shown that the two proteins bind to poly(U)-Sepharose and that homopolynucleotides protect dilute solutions of both inhibitors from inactivation. It is suggested that the presence of a polynucleotide site on alphasarcin is related to its ribonuclease activity. The existence of a similar site on gelonin might be a clue to its yet unknown mechanism of action.     

Improving diagnostic strategies for ovarian cancer in Filipino women using ultrasound imaging and a multivariate index assay

ObjectiveTo evaluate the clinical performance and overall utility of imaging and biomarker assays in discriminating between benign and malignant ovarian masses in a Filipino population.MethodsThis is a prospective cohort study among Filipino women undergoing assessment for an ovarian mass in a tertiary center. All included patients underwent a physical examination before level III specialist ultrasonographic and Doppler evaluation, multivariate index assay (MIA2G), and surgery for an adnexal mass. Ovarian tumors were classified as high-risk for malignancy based on the International Ovarian Tumour Analysis (IOTA) – Logistic Regression 2 (LR2) score. The ovarian imaging and biomarker results were correlated with the reference standard: histological findings.ResultsAmong the 379 women with adnexal masses enrolled in this study, 291 were evaluable with ultrasound imaging, biomarker assays, and histopathological results. The risk of malignancy was higher for women classified as high-risk based on IOTA-LR2 (≥10%). The sensitivity, specificity, and diagnostic accuracy for the prediction of malignancy were 81.2%, 81%, and 0.81 (95% CI: 0.77–0.86) for IOTA-LR2; 77.5%, 66.7%, and 0.72 (95% CI: 0.67–0.77) for CA-125; and 91.3%, 41.2%, and 0.66 (95% CI: 0.62–0.71) for MIA2G. A combination of IOTA-LR2 and MIA2G significantly influenced the diagnostic performance and the result. When MIA2G was combined with IOTA-LR2 in parallel, the sensitivity (94.2%) and NPV (87.7%) increased, but the specificity (37.3%) decreased. When combined with IOTA-LR2 in series, there were fewer false positives, which resulted in improved specificity (85%).ConclusionThis study determined the utility of ovarian imaging and a second-generation multivariate index assay in predicting the risk of ovarian malignancy. IOTA-LR2 and MIA2G were useful in classifying patients with a high risk for ovarian malignancy.     

Inactivation of cytochrome P-450 and production of N-alkylated porphyrins caused in isolated hepatocytes by substituted dihydropyridines. Structural requirements for loss of haem and alkylation of the pyrrole nitrogen atom

Incorporation of 4.5 nmol fluorescein isothiocyanate/mg rabbit sarcoplasmic reticulum, or of 7.4 nmol/mg purified ATPase, was sufficient to inhibit the activity completely. These results are not consistent with the suggestion (Pick, U. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 626, 255-261) that 2 mol ATPase were inhibited by each mole of reagent incorporated. A single labelled peptide was purified from the inhibited ATPase and it was shown that Lys 3/190, 10 residues from the N-terminus of tryptic fragment B, was the reactive lysine residue. This site is close to a potential nucleotide-binding fold in the ATPase sequence. A similar peptide showing only 2 conservative replacements was isolated from the sarcoplasmic reticulum of the lobster.