Validation of gene regulatory networks: scientific and inferential

Gene regulatory network models are a major area of study in systems and computational biology and the construction of network models is among the most important problems in these disciplines. The critical epistemological issue concerns validation. Validity can be approached from two different perspectives (i) given a hypothesized network model, its scientific validity relates to the ability to make predictions from the model that can be checked against experimental observations; and (ii) the validity of a network inference procedure must be evaluated relative to its ability to infer a network from sample points generated by the network. This article examines both perspectives in the framework of a distance function between two networks. It considers some of the obstacles to validation and provides examples of both validation paradigms.     

RBC NOS: regulatory mechanisms and therapeutic aspects

Nitric oxide (NO), one of the most important vascular signaling molecules, is primarily produced by endothelial NO synthase (eNOS). eNOS is tightly regulated by its substrate l-arginine, cofactors and diverse interacting proteins. Interestingly, an NO synthase (NOS) was described within red blood cells (RBC NOS), and it was recently shown to significantly contribute to the intravascular NO pool and to regulate physiologically relevant mechanisms. However, the regulatory mechanisms and clinical implications of RBC NOS are unknown. The aim of this review is to highlight intracellular RBC NOS interactions and the role of RBC NOS in RBC homeostasis. Furthermore, macro- and microvascular diseases affected by RBC-derived NO are discussed.     

The scientific basis of bioassays

The ultimate goal of ecotoxicological testing is to predict ecological effects of chemicals and other stressors. Since damage should be avoided rather than corrected after it occurs, the predictive value of such tests is crucial. A modest base of evidence shows that, in some cases, extrapolations from bioassays on one species to another species are reasonably accurate and, in other cases, misleading. Extrapolations from laboratory bioassays to response in natural systems at the population level are effective if the environmental realism of the bioassay is sufficiently high. When laboratory systems are poor simulations of natural systems, gross extrapolation errors may result. The problem of extrapolating among levels of biological organization has not been given the serious attention it deserves, and currently used methodologies have been chosen for reasons other than scientific validity. As the level of biological organization increases, new properties are added (e.g., nutrient cycling, energy transfer) that are not readily apparent at the lower levels. The measured responses (or end points) will not be the same at all levels of biological organization, making the validation of predictions difficult. Evidence indicates that responses of ecologically complex laboratory systems correspond to predicted and documented patterns in stressed ecosystems. The difficulties of improving the ecological evidence used to predict adverse effects are not insurmountable since the essence of predictive capability is the determination of effects thresholds at all levels of organization. The dilemma between basing predictive schemes on either traditional or holistic methods can only be solved by facing scientific and ethical questions regarding the adequacy of evidence used to make decisions of environmental protection.     

Glucose transport in Achlya: characterization and possible regulatory aspects

The freshwater fungus Achlya transported D-(+)glucose (glucose) and 2-deoxy-D-glucose (deoxyglucose) by an energy-related system. Their transport4 was inhibited by uncouplers of metabolic energy such as 2,4-dinitrophenol, cyanide, azide, and carbonylcyanide-p-chlorophenylhydrazone. Besides inhibiting each other, glucose and deoxyglucose transport was inhibited by D-(+)galactose, D-(+)mannose, and D-(+)xylose. Many other sugars tested failed to inhibit glucose transport implying a certain degree of specificity. Glucose transport was pH (optimum at 6.5) and temperature (optimum at 30-40 degrees C) dependent. Glucose transport was also inhibited by citrate, N6-substituted adenines (cytokinins), and iodine. None of these agents penetrated the cell membrane within the brief (1-3-min) period in which glucose transport was measured. In every case, transport was inhibited within 10 s (the shortest time in which measurements could be made). When cells were osmotically shocked to release a cell-wall membrane phosphorylated proteoglycan (PPG), they became incapable of transporting glucose for several hours until new PPG material was reisolable from the membrane by osmotic-shock treatment. The osmotically shocked cells could not transport glucose or deoxyglucose. No glucose-binding protein was detected in the shock fluid. Practically all of the glucose transported within 1-2 min was recovered as glucose-6-phosphate. No other phosphorylated sugar was detected suggesting that glucose may be phosphorylated in transport. Related studies have shown that citrate removed calcium bound by PPG; N6-substituted adenines were bound by PPG while three polyphosphorylated dinucleosides, HS3, HS2, and HS1, were displaced from it. Iodine formed stable complexes with the HS compounds. All of these agents inhibited glucose transport without entering the cell. It is therefore possible that HS compounds, calcium and PPG may be involved in maintaining the cell membrane in proper form for glucose transport.     

Encystation process of Giardia lamblia: morphological and regulatory aspects

One important step in the life cycle of the pathogenic protozoan Giardia lamblia is the transformation of the proliferative form, the trophozoite, into the non-proliferative cyst. This process, known as encystation, can be triggered in vitro. Morphological analysis showed that during trophozoite-cyst transformation, major changes take place: modification of the protozoan shape, internalization of the flagella, fragmentation of the adhesive disk, and appearance of encystation vesicles (ESVs), which later on fuse with the plasma membrane forming the cell wall. Sites of attachment of these vesicles to the inner portion of the protozoan plasma membrane were observed 6 h after the beginning of the encystation process. These sites were only visible when we used high-resolution scanning electron microscopy to study Giardia surface. In order to analyze the involvement of protein kinases and phosphatases on the encystation process, inhibitors of these enzymes were added to the culture medium, and their effect on the differentiation process was determined using light, immunofluorescence, and electron microscopy. Significant inhibition was observed with LY294002, an inhibitor of PI3 kinase; genistein, an inhibitor of tyrosine kinase; and staurosporine, at concentrations, which inhibit protein kinase C. Okadaic acid, an inhibitor or protein phosphatase, and wortmannin, an inhibitor of PI3K, did not interfere with the encystation process. However, they induced the appearance of large and pleomorphic forms where several nuclei and disorganization of the peripheral vesicles were observed.     

Sodium cotransport systems: cellular, molecular and regulatory aspects

The sodium cotransport systems comprise an important group of transport proteins which are involved in the transport of a variety of organic and inorganic solutes across the cellular membrane of animal cells. These systems play a central role in a wide variety of cellular and biochemical processes. We summarize here the current state of knowledge regarding the variety, structure and regulation of this important group of membrane proteins.     

UVA filters in sun-protection products: regulatory and biological aspects

This review of published in vitro and in vivo studies concerning the biological effects of ultraviolet A (UVA; 320-400 nm) radiation illustrates the evidence for combining UVA and UVB filters in sun-protection products. These data have led to the development of new sunscreens as well as methods to evaluate their efficacy. After listing the UVA filters available and briefly noting the requirements for a high SPF, broad-spectrum sunscreen, the methods for evaluating the level of UVA protection will be described. This article also summarizes several studies looking at the prevention of erythema, pigmentation, DNA damage, photoimmunosuppression, photoaging and photodermatoses. These data demonstrate in vitro and in vivo that only well-balanced UVA-UVB sunscreens, absorbing over the entire UV spectrum are able to prevent or significantly reduce the associated biological damage.     

Phosphoinositide-specific phospholipase C: structural basis for catalysis and regulatory interactions

Phosphoinositide-specific phospholipase C (PI-PLC) isozymes have an important role in cellular responses to a variety of extracellular signals. Recently, the three-dimensional structures of their isolated domains and of the multidomain core, common to all PI-PLCs, have been solved. This provided an insight into the domain organization of PI-PLCs and, together with the structure-function analysis, contributed towards an understanding of the molecular mechanisms of catalysis and regulation.     

The regulatory basis of melanogenic switching

Melanin produced in follicular melanocytes is the major basis for pigmentation of hair and wool in mammals. Two major types of melanin may be synthesized, the black/brown eumelanin and the reddish/yellow pheomelanin. Based on available cell biological evidence and reasonable assumptions, a mathematical model is developed to improve our understanding of melanogenic switching, i.e. the switching between eumelanin and pheomelanin production depending on the extracellular signalling context.In 1993, Ito proposed that melanogenic switching is due to the covalent binding of the intermediate DOPAquinone to the enzyme glutathione reductase. We were only able to obtain a good fit to available experimental data on the relation between pheomelanin levels and the activity of the key enzyme tyrosinase by taking Ito's hypothesis into account. Thus, our results support Ito's hypothesis, and suggest that melanogenic switching may be due to a jump between two stable production pattern states when the tyrosinase activity varies between two bifurcation levels. This implies that small changes in the levels of external regulatory factors may cause an accentuated change in the proportion of the produced colour pigments and may explain the fact that mammalian coat patterns often exhibit sharply delimited patches of either black or reddish colour.     

Fermented pig liquid feed: nutritional, safety and regulatory aspects

Fermented liquid feed has been lately much investigated in order to compensate the use of antibiotics in pig production. The fermentation process has been claimed to be the reason of the benefits associated with this type of feeding. However, contradictory results have been obtained in feeding trials due to the variable conditions in each experiment. This review focuses on the different factors that would ensure a proper fermentation with all its beneficial effects. In particular, while fermenting a liquid diet with lactic acid bacteria has been shown to improve the quality of feed and to be beneficial to the health of the animals, spontaneously fermented liquid feed appears to be unsafe for the pigs and eventually affects the consumers' safety. Consequently, the use of specific starters or inoculants to ensure the proper fermentation could be a practical solution. The regulatory status of fermented liquid feed in the EU is still unclear, but the use of specific inoculants could be considered as a special case of microbial feed additives.     

Derepression of mitochondria and their enzymes in yeast: regulatory aspects

We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate:cytochrome c reductase and NADH:cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for l-malate, l-glutamate, and l₈-isocitrate. Cytochromes b, c₁, and aa₃ are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected.We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4%; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa₃, b, and c₁; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than 6-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression.The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.