Carboxylate anion binding in the cyclohexaamylose cavity: A steric and electronic evaluation

This investigation focuses on the steric and electronic requirements for the binding of carboxylate anions in the cyclohexaamylose cavity. The geometries and thermodynamic stabilities of the 3,5-dimethyl-4-hydroxybenzoic acid, 3,5-dimethyl-4-hydroxycinnamic acid, and 3,5-dimethyl-4-hydroxyhydrocinnamic acid cyclohexaamylose complexes are evaluated at pH = 7.60 and 12.00. Results indicate that the carboxylate anions of 3,5-dimethyl-4-hydroxycinnamic acid and 3,5-dimethyl-4-hydroxyhydrocinnamic acid bind in the cyclohexaamylose cavity at both pH = 7.60 and 12.00. In addition, the dependence of the stability of the resulting complexes on pH is shown to be marginal. These findings suggest that if a carboxylate anion is to bind in the cyclohexaamylose cavity, it must be able to adopt a position in the cavity which allows for at least partial solvation of charge.     

Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation

The use of antibiotic-resistance genes as selectable markers in transgenic organisms is coming under increased scrutiny, for fear that they may spread to human pathogens, thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonas fluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA) to maintain an expression plasmid under control of a repressible promoter and a kanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene. We investigated using auxotrophic markers to replace these two antibiotic resistance genes: pyrF (encoding orotidine-5'-phosphate decarboxylase) in place of tetR/tetA and proC (encoding pyrroline-5-carboxylate reductase) in place of kanR, complementing their respective precise chromosomal deletions created by allele exchange using a suicide vector carrying pyrF as a counterselectable marker. The resulting strains, devoid of antibiotic-resistance genes, were shown to achieve high productivity of nitrilase and thermostable alpha-amylase equal to that of the former antibiotic-resistant production host. The production plasmids were stable. The pyrF (uracil-dependent) background of the production host strain also allows us to sequentially alter the genome to incorporate other desired genomic changes, deletions, or insertions using 5'-fluoroorotic acid counterselection, restoring the selectable marker after each step.     

Mechanisms of reproductive isolation among sympatric broadcast-spawning corals of the Montastraea annularis species complex

Many coral species spawn simultaneously and have compatible gametes, leading to controversy over the nature of species boundaries and the frequency with which hybridization occurs. Three western Atlantic corals, Montastraea annularis, M. faveolata, and M. franksi, typify this controversy; they all spawn sympatrically on the same evenings after the fall full moons. Here we show, in both Panama and the Bahamas for multiple years, how a variety of mechanisms may act in concert to reproductively isolate all three species. Field studies indicate that M. franksi spawns two hours earlier than the other two species, and the eggs released during this earlier period disperse an average of 500 m by the time the other species spawn. Field measures of fertilization indicate that peak fertilization occurs when spawning synchrony is high and that corals that spawn at the tails of the spawning distributions have greatly reduced fertilization success. Laboratory studies indicate that there is a gametic incompatibility between M. faveolata and the other two species. There are regional differences in the gametic compatibility of M. franksi and M. annularis. In Panama, the two species are completely compatible, whereas in the Bahamas, M. franksi sperm can fertilize M. annularis eggs but the reciprocal cross often fails. Gamete age influences patterns of fertilization, such that very young eggs seem resistant to fertilization and old sperm lose viability after two hours. In sum, the combination of temporal differences in spawning, sperm aging, gamete dispersal and dilution, and gametic incompatibility act in various combinations among the three species, making it unlikely that hybrid fertilization would occur.     

The demographics and distribution of type B Niemann-Pick disease: novel mutations lead to new genotype/phenotype correlations

We have collected demographic and/or mutation information on a worldwide sample of 394 patients with type B Niemann-Pick disease (NPD). The disorder is panethnic, with the highest incidence occurring in individuals of Turkish, Arabic, and North African descent. Only five of the 394 patients were Ashkenazi Jewish, revealing that, unlike the type A form of NPD, type B NPD does not occur frequently within this population. Mutation analysis of the acid sphingomyelinase (ASM) gene (designated "SMPD1") was performed on 228 patients (324 unique alleles), and several novel, "common" mutations were found. Among these were the L137P, fsP189, and L549P mutations, which accounted for approximately 75% of the alleles in Turkish patients, the H421Y and K576N mutations, which accounted for approximately 85% of the alleles in Saudi Arabian patients, the S379P, R441X, R474W, and F480L mutations, which accounted for approximately 55% of the alleles in Portuguese/Brazilian patients, and the A196P mutation, which accounted for approximately 42% of the alleles in Scottish/English patients. The previously reported DeltaR608 mutation occurred on approximately 12% of the alleles studied. Overall, a total of 45 novel mutations were found, and several new genotype/phenotype correlations were identified. In particular, the L137P, A196P, and R474W mutations were consistent with a less severe form of type B NPD, whereas the H421Y and K576N mutations led to an early-onset, more severe form that was specific to Saudi Arabia. These data provide the first extensive demographic assessment of this disorder and describe several new mutations that can be used to predict phenotypic outcome and to gain new insights into the structure and function of ASM.     

Monitoring of complex industrial bioprocesses for metabolite concentrations using modern spectroscopies and machine learning: application to gibberellic acid production

Two rapid vibrational spectroscopic approaches (diffuse reflectance-absorbance Fourier transform infrared [FT-IR] and dispersive Raman spectroscopy), and one mass spectrometric method based on in vacuo Curie-point pyrolysis (PyMS), were investigated in this study. A diverse range of unprocessed, industrial fed-batch fermentation broths containing the fungus Gibberella fujikuroi producing the natural product gibberellic acid, were analyzed directly without a priori chromatographic separation. Partial least squares regression (PLSR) and artificial neural networks (ANNs) were applied to all of the information-rich spectra obtained by each of the methods to obtain quantitative information on the gibberellic acid titer. These estimates were of good precision, and the typical root-mean-square error for predictions of concentrations in an independent test set was <10% over a very wide titer range from 0 to 4925 ppm. However, although PLSR and ANNs are very powerful techniques they are often described as "black box" methods because the information they use to construct the calibration model is largely inaccessible. Therefore, a variety of novel evolutionary computation-based methods, including genetic algorithms and genetic programming, were used to produce models that allowed the determination of those input variables that contributed most to the models formed, and to observe that these models were predominantly based on the concentration of gibberellic acid itself. This is the first time that these three modern analytical spectroscopies, in combination with advanced chemometric data analysis, have been compared for their ability to analyze a real commercial bioprocess. The results demonstrate unequivocally that all methods provide very rapid and accurate estimates of the progress of industrial fermentations, and indicate that, of the three methods studied, Raman spectroscopy is the ideal bioprocess monitoring method because it can be adapted for on-line analysis.     

Performance of controlled-release formulations of trimedlure to attract the Mediterranean fruit fly, Ceratitis capitata

Mediterranean fruit flies (Ceratitis capitata Wied.) are detected by traps baited with the attractant, trimedlure (TML), deposited on the standard cotton dental roll dispenser. Since this dispenser is effective for only 2–4 weeks, controlled-release dispensers may extend the field life of TML-baited traps. A polymeric plug containing 70% by weight TML was highly effective for 8–12 weeks. Other formulations were less effective in the tests with released flies. A release rate of ca. 17 mg of standard TML/day or higher gave insect captures which were generally lower but statistically equivalent to those with the reference dispenser which released 61–63 mg/day. Normalized insect catches increased approximately as the square root of the release rate; the curvilinear regression equation suggested that TML release rates of ca. 52 and 15 mg/day give insect captures equal to ca. 100% and 50%, respectively, of the reference catches. The most attractive TML isomer (TML-C) was a highly effective attractant at a release rate of ca. 4 mg/day.

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Résumé C. capitata était attiré par des pièges alimentés avec de la trimedlure (TML), attractif déposé sur un distributeur commercial de coton dentaire en rouleau. Puisque ce distributeur ne fonctionne que 2 à 4 semaines, des distributeurs à libération contrôlée peuvent augmenter le temps de fonctionnement de pièges fournis en TML. Un bouchon en polymère contenant en poids 70% de TML a été très efficace pendant 8 à 12 semaines. D'autres formules ont été moins efficaces avec des mouches lâchées. Un taux de libération, de 17 mg ou plus de TML moyen par jour, capture moins, mais sans différence statistique significative, qu'un distributeur témoin qui libérait 61 à 63 mg/j. Les captures d'insectes ont augmenté à peu près comme le carré du taux de libération; l'équation de régression curviligne a conduit à penser que la libération de TML à des taux de 52 et 15 mg/j, provoque des captures égales à 100% et 50% des captures de référence. L'isomère le plus attractif (TML-C) a été un attractif très efficace au taux de libération de 4 mg/j.

     

Effect of 1,3,6-triaminohexane and 1,4,7-triaminoheptane on growth and polyamine metabolism in SV-3T3 cells treated with 2-difluoromethylornithine

The possibility that one or both of the synthetic triamines, 1,3,6-triaminohexane and 1,4,7-triaminoheptane, could substitute for the naturally occurring polyamines in the growth of SV-3T3 cells was investigated. It was found that these triamines did lead to a restoration of growth in cells in which spermidine content had been depleted by exposure to the ornithine decarboxylase inhibitor 2-difluoromethylornithine. This resumption of a normal growth rate occurred prior to the reduction in the content of cellular decarboxylated S-adenosylmethionine, suggesting that this nucleoside (which increases in concentration several hundred-fold in cells treated with 2-difluoromethylornithine) does not cause the reduction of cell growth. However, unlike the increase in cell growth brought about by spermidine, which continued indefinitely, the increase produced by 1,3,6-triaminohexane or 1,4,7-triaminoheptane was transient. Cell growth in the presence of 2-difluoromethylornithine and these triamines stopped after about three or four population doublings. This corresponded to the time at which the intracellular spermine content of the cells was reduced to values less than 20% of normal. It is suggested that the increased growth rate of spermidine-depleted cells in response to these triamines is due to their uptake into the cell and ability to displace spermine from intracellular sites, thus making spermine available to fulfill the polyamine function(s) essential for growth. These results indicate that the naturally occurring polyamines spermidine or spermine are essential for continued cell growth and cannot be replaced by analogues containing only primary amino groups.     

An approach to the study of structure-function relationships of MHC class I molecules: isolation and serologic characterization of H-2Kb somatic cell variants

Somatic cell variants expressing an altered antigenic form of the H-2Kb molecule were isolated for the purpose of performing structure-function analysis of a class I MHC molecule. Over 25 independently isolated variants were derived from an Abelson virus transformed pre- B cell line (R8) by mutagenesis with ethyl methane sulfonate or ethyl nitrosourea. Negative selection was performed by complement-dependent cytotoxicity with anti-H-2Kb monoclonal antibodies subsequently followed by positive selection to separate the H-2Kb surface negative variants from structural variants. Biochemical characterization of a random selection of three independent variants indicated that the variant H-2Kb molecule was present in normal amounts in lysates, and was unchanged in size. Cytofluorometric analysis with the use of a panel of seven monoclonal antibodies against H-2Kb indicated that all of the variants had lost one or more alloantigenic determinants (monoclonal antibody binding sites). For these variants, the pattern of monoclonal antibody loss of recognition suggested that antibody defined alloantigenic determinants appear to be discretely localized to a single domain, either the alpha 1 or the alpha 2 domain, of the H-2Kb molecule. In contrast, CTL recognition of the Kb molecule of these variants depends on involvement of both alpha 1 and alpha 2 domains as shown in the companion paper.     

In vivo analysis of the plasmid pAM beta 1 resolution system.

The promiscuous plasmid pAM beta 1 from Gram-positive bacteria encodes a resolution system which differs from that of Tn3 in that (i) it requires a histone-like protein and an unusual resolvase-DNA interaction to promote recombination and (ii) it mediates in vivo DNA inversion in plasmid substrates. In this in vivo analysis, the pAM beta 1 resolution site is narrowed down to a 99 bp segment, the strand exchange is mapped within 10 bp and the serine residue at position 10 of the resolvase is shown to be essential for enzyme activity. In addition, data showing that the resolution system does not promote DNA inversion in the Bacillus subtilis chromosome are presented. Implications of this observation are discussed.