On the interaction between stabilizing social factors and destabilizing trophic factors in small rodent populations

Mathematical models are developed in order to analyze whether or not social factors, such as, for example, the “social fence” (J. B. Hestbeck, 1982, Oikos 39, 157–163) will stabilize population density: the dynamic interaction between social factors and (dynamic) trophic factors is analyzed. It is concluded that social factors such as the “social fence” tend to stabilize population density; hence, if density cycles (as, e.g., seen in many microtine rodents) are observed in nature, it seems reasonable to conclude that density cycles are driven by, for example, trophic interactions and not by social factors. It is suggested that the “social fence” may explain why so many populations including several microtine populations have fairly stable densities despite the ever-existing destabilizing trophic interactions. Contrary to what is implied by J. B. Hestbeck (1983, “A Mathematical Model of Population Regulation in Cyclic Mammals,” Lecture notes in biomathematics, Vol. 52, Springer-Verlag, Berlin/New York), the analysis presented in this paper demonstrates that seasonal environmental changes are not essential for the generation of regular density cycles. Seasonal changes may, however, be necessary for generating a microtine-like density cycle. Empirical information on microtine rodents relating to the “social fence hypothesis” is discussed.     

Information integration of protein–protein interactions as essential tools for immunomics

After a brief introduction to point out the necessity to advance for a global understanding of the macromolecular interactions occurring during the immune system development and responses, Section 2 will be devoted to analyse the current tools for an automatic location of information on these protein–protein interactions in the web. In the next section (Section 3), we will point out different action lines to improve these tools and, consequently, to increase the efficiency to establish (to understand) the “protein network skeleton” that controls our immune responses. Finally, we will briefly present our current strategy and work to advance towards this goal.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

Spatial and temporal preferences for trans-splicing in Ciona intestinalis revealed by EST-based gene expression analysis

Ciona intestinalis is a useful model organism to analyze chordate development and genetics. However, unlike vertebrates, it shares a unique mechanism called trans-splicing with lower eukaryotes. In the computational analysis of trans-splicing in C. intestinalis we report here, we discovered that although the amount of non-trans-spliced and trans-spliced genes is usually equivalent, the expression ratio between the two groups varies significantly with tissues and developmental stages. Among the seven tissues studied, the observed ratios ranged from 2.53 in “gonad” to 19.53 in “endostyle”, and during development they increased from 1.68 at the “egg” stage to 7.55 at the “juvenile” stage. We further hypothesize that this enrichment in trans-spliced mRNAs in early developmental stages might be related to the abundance of trans-spliced mRNAs in “gonad”. Our analysis indicates that in C. intestinalis, although there may not exist strong fundamental requirements for genes to be trans-spliced, the populations of non-trans-spliced and trans-spliced genes are likely to be spatially and temporally regulated differently.     

Spatial organization of interphase chromosomes and the role of chromatin fibril dynamics in the positioning of genome elements

Many studies are devoted to the analysis of interphase chromosome architecture due to the evidence of the functional-dependent spatial organization of the genome. These studies are based on classical cytological methods, as well as on biochemical approaches (3C, 4C, 5C, Hi-C), which allow one to detect long-range interactions between fragments of chromatin fibril, including the genome-wide interactions. In this review, we discuss the results of these projects, which allow us to explain the functional basis of nucleus multilevel compartmentalization and to identify the principles of high-level chromatin organization. Special attention is paid to the enhancer-promoter interactions, which are important for the regulation of gene expression. In this regard, we provide a new interpretation to the model of an active chromatin hub and to the alternative model of an active chromatin compartment, which was proposed during reconsideration of some steps of the 3C procedure.     

Comparison of the Antimicrobial Adhesion Potential of Human Body Fluid Glycoconjugates Using Fucose-Binding Lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus Lectin (UEA-I)

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are “secretors” express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of “secretors” and “nonsecretors” with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that “nonsecretor” body fluids were not less efficient than those of “secretors” in PA-IIL blocking. UEA-I, which interacted only with the “secretors” glycoproteins, was most sensitive to those of the seminal fluids.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

An evolutionary-network model reveals stratified interactions in the V3 loop of the HIV-1 envelope

The third variable loop (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope is a principal determinant of antibody neutralization and progression to AIDS. Although it is undoubtedly an important target for vaccine research, extensive genetic variation in V3 remains an obstacle to the development of an effective vaccine. Comparative methods that exploit the abundance of sequence data can detect interactions between residues of rapidly evolving proteins such as the HIV-1 envelope, revealing biological constraints on their variability. However, previous studies have relied implicitly on two biologically unrealistic assumptions: (1) that founder effects in the evolutionary history of the sequences can be ignored, and; (2) that statistical associations between residues occur exclusively in pairs. We show that comparative methods that neglect the evolutionary history of extant sequences are susceptible to a high rate of false positives (20%–40%). Therefore, we propose a new method to detect interactions that relaxes both of these assumptions. First, we reconstruct the evolutionary history of extant sequences by maximum likelihood, shifting focus from extant sequence variation to the underlying substitution events. Second, we analyze the joint distribution of substitution events among positions in the sequence as a Bayesian graphical model, in which each branch in the phylogeny is a unit of observation. We perform extensive validation of our models using both simulations and a control case of known interactions in HIV-1 protease, and apply this method to detect interactions within V3 from a sample of 1,154 HIV-1 envelope sequences. Our method greatly reduces the number of false positives due to founder effects, while capturing several higher-order interactions among V3 residues. By mapping these interactions to a structural model of the V3 loop, we find that the loop is stratified into distinct evolutionary clusters. We extend our model to detect interactions between the V3 and C4 domains of the HIV-1 envelope, and account for the uncertainty in mapping substitutions to the tree with a parametric bootstrap.     

Cambrian view

The analysis of visual systems is a valuable method of assessing phylogenetic processes. As in the present animal world, we find simple and complex systems in the Lower Cambrian. One may detect “simple eyes” for example with an advanced design in lobopodians, while the existence of even more simple “simple eyes” is very probable but still to be proved. More complex systems are to be found. In Leanchoilia illecebrosa Hou, 1987 and Leanchoilia superlata Walcott, 1912 there are probable dorsal median eyes and a pair of fine, stalked ventral eyes. Both systems may contribute to phylogenetic and systematic discussions. These presumably movable stalked eyes may be regarded as an adaptation to a mobile lifestyle. They suggest that the physiologic principle of nystagmus to stabilise the visual world of an animal in motion was already realised in Leanchoilia, perhaps for the first time. To analyse the surface of the early eyes from the Lower Cambrian – not only of Leanchoilia, but of any other forms as well – the number, shape and other parameters of the lenses could lead to further knowledge regarding vision in early invertebrates.     

A cultivar comparison of plant regeneration from suspension cells,callus, and cormel slices ofGladiolus

Callus was initiated from either cormel slices or in vitro-grown plants of sixGladiolus cultivars cultured on Murashige and Skoog’s basal salts medium supplemented with either 10 mg/liter (53.8 µM) 1-naphthaleneacetic acid, 2 mg/liter (9.3 µM) dicamba, or 0.5 mg/liter (2.2 µM) 2,4-dichlorophenoxyacetic acid. More plants were regenerated from callus of the cultivar “Peter Pears” as compared to “Jenny Lee,” “Florida Flame,” or “Golden Year.” No plants were regenerated from callus of “Rosa Supreme” or “Purity White.” Plants were regenerated from 2 and 6-mo.-old suspension cells of “Jenny Lee” and “Peter Pears” but not from “Florida Flame.” Cormel slices cultured on Murashige and Skoog’s basal salts medium supplemented with 1 mg/liter (4.4 µM) 6-benzylaminopurine regenerated plants from all six cultivars indicating a cultivar-independent system of plant regeneration.     

Global Quantitative Modeling of Chromatin Factor Interactions

Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the “chromatin codes”) remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles — we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions.     

Formamide denaturation of chromosomal DNA for in situ hybridization and C-band preparation in the guinea pig, Cavia porcellus

Cytological preparations were incubated in 0.07 N NaOH at room temperature or 90% formamide (final salt concentration 2 × SSC) at either 65 °C or 37 °C for 2.5 h to denature guinea pig chromosomes. Chromosomes treated with NaOH or formamide at 65 °C showed a large amount of DNA loss, while chromosomes treated with formamide at 37 °C showed little or no DNA loss. Repeated sequences were isolated from guinea pig DNA and [³H]cRNA was transcribed with Escherichia coli RNA polymerase for in situ hybridization. Localization of the [³H]cRNA occurred in the centromeric regions and C-band positive short arms of almost all of the chromosomes in the NaOH preparations. Chromosomes treated with formamide at 65 °C showed the same grain distribution with a decrease in the number of grains/cluster. Slides incubated in formamide at 37 °C showed localization in only a few chromosomes and the number of grains/cluster was greatly diminished. Thermal denaturation of isolated chromatin indicated that incubation of chromosomes in formamide at 37 °C did not fully denature the DNA. C-bands could be induced by treating slides in formamide at either 65 °C or 37 °C when followed by a “reassociation” in 2 × SSC at 65 °C for 16 h. If the “reassociation” step was omitted, C-bands were found in the 65 °C formamide slides but not the 37 °C formamide slides.     

Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer

The aim of this work was the development of rapid methods suitable for monitoring the growth of the oleaginous yeast Waltomyces lipofer by means of cell size, vitality and the development of internal lipid droplets throughout different growth phases. Oleaginous yeasts are of interest for the industrial production of lipids and therefore precise monitoring of growth characteristics is needed.This paper provides information about both the method development as well as about examples for their use in monitoring applications. Cell size and shape were determined using FPIA (Flow Particle Image Analysis). Vitality and internal lipid droplets were measured using two independent staining methods for Flow Cytometry. Double staining with cFDA & PI was used for the distinction between “vital”, “sublethal” and “dead” subpopulations, whereas Nile Red allowed the monitoring of lipid accumulation. In this approach the method for vitality measurement was optimized focussing on the staining buffer. An addition of 25 mM citric acid and pH 4.8 revealed to be optimal. The cells in the growth experiment showed a constantly high vitality, which was always above 90%, but slowly decreasing over time. In the course of lipid droplet development it could be seen that the cell size and the Nile Red fluorescence intensity increased. It was demonstrated that the tested method combination provides a powerful tool for rapid fermentation monitoring of the oleaginous yeast W. lipofer, which allows gaining information about the desired growth characteristics in less than 45 min. Further applications for the two methods will be discussed in this article.     

Yeast sterol C24-methyltransferase: role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis

The role of Try-81 in the reaction catalyzed by Saccharomyces cerevisiae sterol 24-C-methyltransferase (Erg6p) was investigated kinetically and for product differences against a panel of position-81 mutants in which Tyr was substituted with Trp, Phe, Ile, Leu, Val and Ala. The residue chosen for mutation is one that was reported previously to accept fecosterol and yield a set 24-ethyl (idene) sterol products typical of plants, showing the amino acid residue is located close to the transient C25 carbocation intermediate in the active site. One group of mutants (aromatic) tested with the natural substrate zymosterol accelerated the C-methylation reaction (k_cat/K_m) whereas the other group of mutants (aliphatics) decreased catalytic competence as the amino acid side chain was downsized. Mutating to aromatic and assaying with the substrate analog designed as a suicide substrate 26,27-dehydrozymosterol favored C26-monol formation, whereas mutating to the aliphatic of smaller size favored C26-diol formation (a measure of enzyme alkylation). In no case was zymosterol converted to an intermediate that formed a C25-diol. Thermodynamic analysis (determination of E_a, ΔG^‡, ΔH^‡ and TΔS^‡) for the C-methylation reaction performed by these enzymes assayed with the substrate and its analog or zymosterol paired with the “charged’ high energy intermediate (HEI) analogs 24(R,S)25,epiminolanosterol and 25-azalanosterol or “neutral” membrane insert ergosterol showed that mutation to aromatics can reduce inhibitor potency (measured as K_m/K_i), yet catalysis can improve in Trp81 by the introduction of a gain in free energy associated with stabilization of the transition state of a rate-controlling step directed toward turnover. Alternatively, mutation to the smaller aliphatic amino acid side chains led to a destabilization in the active site structure which was accompanied by increases in the partition ratios associated with abortive complex formation. The results are explained by consideration of the functional differences attributed to Tyr81 substitution to aromatics and aliphatics of different size involved with cation-π or hydrogen bonding interactions and in the activation barriers required of differing side chain conformations to orient the reactants in the direction of turnover versus enzyme inactivation.