PSAMM: A Portable System for the Analysis of Metabolic Models

The genome-scale models of metabolic networks have been broadly applied in phenotype prediction, evolutionary reconstruction, community functional analysis, and metabolic engineering. Despite the development of tools that support individual steps along the modeling procedure, it is still difficult to associate mathematical simulation results with the annotation and biological interpretation of metabolic models. In order to solve this problem, here we developed a Portable System for the Analysis of Metabolic Models (PSAMM), a new open-source software package that supports the integration of heterogeneous metadata in model annotations and provides a user-friendly interface for the analysis of metabolic models. PSAMM is independent of paid software environments like MATLAB, and all its dependencies are freely available for academic users. Compared to existing tools, PSAMM significantly reduced the running time of constraint-based analysis and enabled flexible settings of simulation parameters using simple one-line commands. The integration of heterogeneous, model-specific annotation information in PSAMM is achieved with a novel format of YAML-based model representation, which has several advantages, such as providing a modular organization of model components and simulation settings, enabling model version tracking, and permitting the integration of multiple simulation problems. PSAMM also includes a number of quality checking procedures to examine stoichiometric balance and to identify blocked reactions. Applying PSAMM to 57 models collected from current literature, we demonstrated how the software can be used for managing and simulating metabolic models. We identified a number of common inconsistencies in existing models and constructed an updated model repository to document the resolution of these inconsistencies.     

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitate the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.     

A RECONSTRUCTION OF CELL DEVELOPMENT IN THE SHOOT APEX OF MAIZE

Somatic mutations were induced in maize embryos in order to follow the albino-tissue patterns in mature plants. A reconstruction of cellular development in the shoot apex has been attempted. Two strains of maize were employed, wd/Yg₂ and pastel-8549/y₁ for seed irradiation with gamma rays. After mature plants had developed from this radiated seed, the sectored plants were analyzed in detail for their patterns of albino tissue. The location and frequency of these patterns were correlated with cell number at various sites of the initial shoot apex in order to deduce the number of cells contributing to each frequency class. Various lines of evidence lead to the conclusion that the cellular differentiation in the shoot apex is organized and a relatively stable process. Apparently a few cells in the apical dome provided daughter tissue for the upper half of the maize plant. Various sector patterns are diagrammed and the position of their albino tissue is explained in relation to the location of a specific cell in the apex.     

INCORPORATION OF H3-THYMIDINE INTO CHLOROPLAST DNA OF MARINE ALGAE

The chloroplasts of three genera of marine algae, Dictyota, Padina, and Bryopsis, were labeled with tritiated-thymidine for various time periods during culture in "Erd-Schreiber's" solution. Autoradiographs were prepared from both smeared and sectioned material. They revealed that almost all of the radioactivity was in the cytoplasm and associated with the chloroplasts, as detected in the overlying silver halide crystals. Deoxyribonuclease, ribonuclease, and hot trichloracetic acid treatments indicated that the loss of radioactivity corresponded to the removal of DNA and not RNA. Quantitative studies of silver grain distribution suggested that the radioactivity of the labeled DNA originated from the edge of the pyrenoids on either side in the longitudinal direction of Bryopsis chloroplasts. Nuclei did not incorporate H³-thymidine even though cells were dividing rapidly in the three genera examined. It is postulated that the enzyme, thymidine kinase, is absent as a coding sequence of nuclear DNA in algae, but is present in chloroplast DNA. When the chloroplasts of Dictyota and Padina in various stages of division were scored for labeling, there appeared to be a DNA synthesis period, analogous to S period in cell division. This chloroplast-labeling period occurred just previous to fission. Many of the criteria seem to have been satisfied to establish the self-reproducing and semi-autonomous nature of chloroplasts, especially when combined with the chemical, genetic, and morphological evidence.     

Ventilation and oxygen consumption in the hagfish, Myxine glutinosa L.

Ventilation was measured directly in the hagfish, Myxine glutinosa L., by means of an electro-magnetic blood flowmeter. Ventilatory flow and frequency increased from 0.86 ± 0.27 ml·min^?, and 18.2 ± 5.1·min^?, respectively, at 7°C to 1.70 ± 0.20 ml·min^?, and 70.1 ± 9.5·min^? at 15 ·C.Standard oxygen consumption,V?O₂, was measured in non-buried hagfish. V?O₂ was 0.57 ± 0.17μl O₂·g^?1·min^?1 at 7°C, and 0.85 ± 0.12μl O₂·g^?1·min^?1 at 15°C.     

Localization of zein genes in maize

The band patterns of zein polypeptides were determined for many commercial inbred corn lines and maize stocks using isofocusing in agarose gels and sodium dodecyl sulfate (SDS)-urea gels. Each inbred line or homozygous maize strain genotype has a distinct zein profile which has been catalogued according to the distance of charge migration and molecular weight (kilodaltons). Several zein polypeptides were mapped to chromosomes 4 and 10 with the use of reciprocal translocations. The mapping of at least two polypeptides on distal 4L and 10L had not been previously reported. The general methods used in the present research will permit the mapping of all the zein polypeptides to chromosomal sites.Pioneer Hi-Bred International provided financial support.     

Chromosomal sites of integration of simian virus 40 DNA sequences mapped by in situ hybridization in two transformed hybrid cell lines.

The neutralizing characteristics of monoclonal antibodies directed to four antigenic sites on the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus were determined. Neutralization by each antibody resulted in a persistent fraction of nonneutralized virus which varied from 1 to 17% depending on the hemagglutinin-neuraminidase site recognized, but not on the antibody. The addition of antibodies to all four sites on the hemagglutinin-neuraminidase glycoprotein was required to give a level of neutralization comparable with that obtained with polyclonal mouse antiserum. The high persistent fractions were not due to viral aggregates, a high level of variants in the virus stock, the use of insufficient antibody, low antibody avidity, or an effect peculiar to the use of the chicken cells as host. The addition of rabbit anti-mouse immunoglobulin to the persistent fraction left by any of the antibodies resulted in a further reduction in infectivity, often by as much as two logs. Thus, some viral particles are capable of binding antibody while retaining their infectivity. The implications of these findings to the mechanism of neutralization are discussed.     

Generation of a Family-specific Phage Library of Llama Single Chain Antibody Fragments That Neutralize HIV-1

Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.     

CHANGES IN SENSITIVITY OF MAIZE CHROMOSOMES TO X RAYS DURING SEED GERMINATION

Latterell , Richard L., and Dale M. Steffensen . (Brookhaven Natl. Lab., Upton, L. I.. N. Y.) Changes in sensitivity of maize chromosomes to X rays during seed germination. Amer. Jour. Bot. 49(5): 472–478. Illus. 1962.—Changes in the radiosensitivity of maize seeds during early stages of germination were studied by means of somatic-mutation techniques. Seeds heterozygous for the yg₂ (yellow-green) locus were irradiated with 800 r of X rays after soaking in running tap water up to 42 hr. Yellow-green sectors, representing mutations affecting the dominant yg₂ locuss in leaves 4 and 5 of seedling plants were used as a criterion of radiosensitivity. The frequency of somatic sectors was virtually nil for dry seed and for seeds soaked up to 16 hr. Sector frequencies underwent a marked (9- to 15-fold) rise from 16 to 28 hr, reached a plateau of sensitivity and subsequently declined. Manometric studies were conducted on seeds soaked under the same conditions as those irradiated. The rate of oxygen consumption rose rapidly from 0 to 7 hr, remained essentially constant from 7 to 16 hr, then underwent an approximately 2-fold increase from 16 to 24 hr, after which the rate of progressive increase was retarded. The fact that the marked rise in frequency of X-ray-induced somatic sectors coincided with the major increase in oxygen consumption suggests that radiosensitivity of soaked seeds is conditioned by metabolic changes during seed development. Changes in radiosensitivity that followed attainment of peak sector frequencies were apparently governed by factors that influenced the rate of seed development.