Mixed monolayers of linear gramicidins and phospholipid. Surface pressure and surface potential studies.

The behavior of two gramicidins incorporated into lipid monolayers is analyzed on the basis of the force and surface potential area curves. It is shown that the position of the gramicidins (helical axis parallel or perpendicular to the interface) depends on the monolayer pressure and that these molecules are not miscible with dioleoylphosphatidylcholine. Surface potential measurements suggest the existence of a relationship between the single channel characteristics and the surface potential and indicate that the tryptophans are essential for lowering the lipid surface potential in agreement with the single channel behaviour of both gramicidin A and gramicidin M.     

Analysis of disease-associated ND4 mutations: How do we know which mutation is pathogenic?

It is not uncommon to identify more than one mtDNA replacement mutations in the specimens from patients. However, we usually do not know if the identified mtDNA mutation is pathogenic or not. Even functional assays are available to use, we would not know which mutation(s) is to be tested. To provide a rapid method for initial evaluation for the pathogenicity of the replacement mutation, we compared three evolutional analyses: primate conservation index (PCI), mammalian conservation index (MCI), and conservation index across a wide spectrum of species (CI). After analyzing 35 so-called diseases-associated replacement mutations of ND4, we found 8 pathogenic mutations, 15 nonpathogenic mutations, and 12 mutations of undetermined significance. The MCI classification appears to be the best one among the three systems. This study demonstrates that evolutional analysis can serve as a rapid evaluation for the pathogenicity of mtDNA replacement mutations.     

The effects of reaction conditions on the production of γ-cyclodextrin from tapioca starch by using a novel recombinant engineered CGTase

A novel mutant enzyme namely H43T CGTase can produce up to 39% γ-cyclodextrin (γ-CD) compared to the native enzyme which produces only 10% γ-CD. The effect of the reaction conditions on γ-CD production was studied using this mutant CGTase. The effects of substrate–buffer combination, starch pretreatment and concentration, pH, additives and finally the use of a debranching enzyme improved the γ-CD ratio further. The tapioca–acetate pair gave the highest conversion (16% conversion) among four types of starch and four buffer system combinations. Gelatinized starch was preferred compared to raw tapioca starch in producing a high percentage of γ-CD and conversion rate. Higher pH especially pH 8–9 led to a higher proportion of γ-CD, and was relatively more apparent when the concentration of starch was increased. Forty-six percent γ-CD was produced using 2.5% gelatinized tapioca starch at pH 8. Pullulanase enzyme was found to be useful in reducing the viscosity of tapioca starch paste thus increasing the efficiency of utilization of starch by CGTase by at least 20- to 30-fold. Up to 48% γ-CD can be produced when 4% pullulanase-pretreated tapioca starch was reacted with the CGTase mutant. It was also found that the supplementation of the reaction mixture with glucose, toluene, or cyclododecanone improved the γ-CD yield by 42.2, 46.4, 43.4, and 43.4%, respectively. All the parameters involved have been shown to affect the product specificity of the mutant H43T CGTase transglycosylation mechanism.     

Effectiveness of protein baits on melon fly and oriental fruit fly (Diptera: Tephritidae): attraction and feeding

Attraction and feeding responses of oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, Bactrocera cucurbitae (Coquillett), were determined for different protein baits. In separate choice attraction assays for each species, significantly more flies arrived at stations with bait than water, but no differences existed among baits of GF-120 Fruit Fly Bait, GF-120 NF Naturalyte Fruit Fly Bait, Provesta 621 autolyzed yeast extract, and Mazoferm E802. In comparison with B. dorsalis, B. cucurbitae had 2.8 times more responders and a 4.8 times better discrimination between baits and water. In a second attraction assay with only B. dorsalis, volume of bait was negatively correlated to numbers of flies alighting on the bait. Feeding assays for both species demonstrated that time spent feeding and duration on a leaf were both significantly affected by bait type. B. dorsalis fed the longest on Provesta 621, with significantly less feeding on the other baits, and with all baits resulting in more feeding than water. The longest feeding times for B. cucurbitae resulted with Mazoferm E802 and Provesta 621, and all baits except GF-120 NF resulted in eliciting a significantly longer feeding duration than water. In separate toxicology assays for each species, significantly higher mortality resulted from bait formulations containing spinosad compared with blank baits, but no differences existed between GF-120 and GF-120 NF formulations. The differences are discussed between the two Bactrocera species primarily in regard to bait preference, extent of response, and previous work on laboratory flies.     

Novel bait stations for attract-and-kill of pestiferous fruit flies

A novel, visually-attractive bait station was developed in Hawaii for application of insecticidal baits against oriental fruit fly, Bactrocera dorsalis (Hendel), melon fly, Bactrocera cucurbitae (Coquillett), and Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (all Diptera: Tephritidae). The bait station developed represents a supernormal visual stimulus of papaya foliage and takes advantage of the flies' strong response to the high light-reflecting properties of yellow color and of their need for shelter, while fully protecting the bait against rainfall. Field studies revealed that the behavioral response of female fruit flies, in particular C. capitata and B. cucurbitae , to yellow-painted bait stations sprayed with GF-120 NF Naturalyte Fruit Fly Bait was significantly enhanced compared with similarly sprayed bait stations that mimicked the green color of fully grown papaya leaves. Field studies conducted with B. cucurbitae indicated that the period of bait attractiveness can be extended for at least 1 week after bait application due to the rain-fastness properties of the bait stations and the use of a visually-attractive color. Our studies provide the behavioral basis for the development of improved attract-and-kill bait stations for fruit flies in Hawaii. These devices also provide a standardized way of evaluating bait spray formulations, thus allowing for proper comparisons over time, across species, and among geographical areas.     

Physicochemical characterization of chitin and chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and the yields were 30.0-32.2% with that of chitosan C120 being the highest. The degree of N-deacetylation of chitosans (83.3–93.3%) increased but the average molecular weight (483–526 kDa) decreased with the prolonged reaction time. Crab chitosans showed lower lightness and WI values than purified chitin, chitosans CC and CS but higher than crude chitin. With the prolonged reaction time, the nitrogen (8.9–9.5%), carbon (42.2–45.2%) and hydrogen contents (7.9–8.6%) in chitosans prepared consistently increased whereas N/C ratios remained the same (0.21). Crab chitosans prepared showed a melting endothermic peak at 152.3–159.2 °C. Three chitosans showed similar microfibrillar crystalline structure and two crystalline reflections at 2θ = 8.8–9.0° and 18.9–19.1°. Overall, the characteristics of three crab chitosans were unique and differed from those of chitosan CC and CS as evidenced by the element analysis, differential scanning calorimetry, scanning electron microscopy and X-ray diffraction patterns.     

Mode of Binding of the Tuberculosis Prodrug Isoniazid to Heme Peroxidases: BINDING STUDIES AND CRYSTAL STRUCTURE OF BOVINE LACTOPEROXIDASE WITH ISONIAZID AT 2.7 ? RESOLUTION*

Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe⁴²² O, Gln⁴²³ O^ϵ1, and Phe²⁵⁴ O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe³⁺, His¹⁰⁹ N^ϵ2, and Gln¹⁰⁵ N^ϵ2. The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C^β and C^γ atoms of Glu²⁵⁸, and C^γ and C^δ atoms of Arg²⁵⁵. The binding studies indicate that INH binds to LPO with a value of 0.9 × 10⁻⁶ m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H₂O₂ with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.     

Genome-wide detection and analysis of homologous recombination among sequenced strains of Escherichia coli

Background  

Comparisons of complete bacterial genomes reveal evidence of lateral transfer of DNA across otherwise clonally diverging lineages. Some lateral transfer events result in acquisition of novel genomic segments and are easily detected through genome comparison. Other more subtle lateral transfers involve homologous recombination events that result in substitution of alleles within conserved genomic regions. This type of event is observed infrequently among distantly related organisms. It is reported to be more common within species, but the frequency has been difficult to quantify since the sequences under comparison tend to have relatively few polymorphic sites.     

Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis

Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.