Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism.

The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in Escherichia coli and compare favourably with in silico prediction by flux balance analysis.

Extent of incorporation of hydroxycinnamaldehydes into lignin in cinnamyl alcohol dehydrogenase-downregulated plants.

Down-regulation of cinnamyl alcohol dehydrogenase leads to an accumulation of cinnamaldehydes available for incorporation into the developing lignin polymer. Using electron spin resonance spectroscopy we have demonstrated that the parent radical of 4-hydroxy-3-methoxycinnamaldehyde is generated by peroxidase catalysed oxidation. The extent of radical generation is similar to that of 4-hydroxy-3-methoxycinnamyl alcohol and is increased by further aromatic methoxylation. From the distribution of the electron-spin density, it was predicted that the regiochemistry of 4-hydroxy-3-methoxycinnamaldehyde coupling would be similar to that of the corresponding alcohol, with the possibility of a higher degree of 8-O-4 linkages occurring. These predictions were confirmed by polymerisation studies, which also showed that after radical coupling the alpha,beta-enone structure was regenerated. This suggests that, although the cross-linking and physical properties of cinnamaldeyde rich lignins differ from that of normal lignins, cinnamaldehydes are incorporated into the lignin polymer under the same controlling factors as the cinnamyl alcohols.

Effect of phenolic structures on the degradability of cell walls isolated from newly extended apical internode of tall fescue (Festuca arundinacea Schreb.).

Apical internodes of tall fescue (Festuca arundinacea Schreb. var. Clarine) harvested at flowering were sectioned into 5 or 10 equal parts to study in situ degradability and cell wall composition, respectively. The basal (youngest) section had the greatest primary wall content. Cell walls in the upper (older) sections had the highest xylose/arabinose ratio and lignin content and a lignin rich in syringyl units, all typical of extensive secondary wall development. Almost all of the p-coumaric (p-CA) and about half of the ferulic acid (FA) were released by 1 M NaOH and presumed to be ester-linked. The total FA content was approximately double that of p-CA in all sections other than the youngest with a distribution similar to that of total p-CA. However, the ratio of esterified to ether and ether plus ester linked (Et & Et+Es) FA differed with age. Whereas the esterified form remained essentially constant ( approximately 4.5 g/kg of cell wall), Et & Et+Es ferulate increased with increasing age of the tissue and was significantly related to lignin deposition (r = 0.79, P < 0.01). The extent of cell wall degradation after 48 h of incubation in the rumen was inversely related to maturity, falling from 835 g/kg of dry matter in the youngest section to 396 g/kg in the oldest. Both the rate and extent of cell wall degradation were significantly negatively related to the ratio of xylose to arabinose, lignin content, proportion of syringyl units present in lignin, and concentration of Et & Et+Es FA present. A positive relationship between Et & Et+Es FA was also found, with the rate (P < 0.01) being better correlated than the extent (P < 0.05) of cell wall degradation. Application of the newly extended internode model to fescue produced results consistent with the view that both the lignin content and the extent to which lignin was covalently bound to the other wall polymers crucially influenced the rate and extent of degradation.

Dithiocarbamate toxicity toward thymocytes involves their copper-catalyzed conversion to thiuram disulfides, which oxidize glutathione in a redox cycle without the release of reactive oxygen species.

We have reported previously that diethyldithio-carbamate (DDC) and pyrrolidine dithiocarbamate (PDTC) induce apoptosis in rat thymocytes. Apoptosis was shown to be dependent upon the transport of external Cu ions into the cells and was accompanied by the oxidation of intracellular glutathione, indicating the inducement of pro-oxidative conditions (C. S. I. Nobel, M. Kimland, B. Lind, S. Orrenius, and A. F. G. Slater, J. Biol. Chem. 270, 26202-26208, 1995). In the present investigation we have examined the chemical reactions underlying these effects. Evidence is presented to suggest that dithiocarbamates undergo oxidation by CuII ions, resulting in formation of the corresponding thiuram disulfides, which are then reduced by glutathione, thereby generating the parent dithiocarbamate and oxidized glutathione (glutathione disulfide). Although DDC and PDTC were found to partially stabilize CuI ions, limited redox cycling of the metal ion was evident. Redox cycling did not, however, result in the release of reactive oxygen species, which are believed to be scavenged in situ by the dithiocarbamate. DDC and PDTC were, in fact, shown to prevent copper-dependent hydroxyl radical formation and DNA fragmentation in model reaction systems. The thiuram disulfide disulfiram (DSF) was found to induce glutathione oxidation, DNA fragmentation, and cell killing more potently than its parent dithiocarbamate, DDC. Of particular importance was the finding that, compared with DDC, the actions of DSF were less prone to inhibition by the removal of external copper ions with a chelating agent. This observation is consistent with our proposed mechanism of dithiocarbamate toxicity, which involves their copper-catalyzed conversion to cytotoxic thiuram disulfides.

Experimental analysis of large belief networks for medical diagnosis.

We present an experimental analysis of two parameters that are important in knowledge engineering for large belief networks. We conducted the experiments on a network derived from the Internist-1 medical knowledge base. In this network, a generalization of the noisy-OR gate is used to model causal independence for the multivalued variables, and leak probabilities are used to represent the nonspecified causes of intermediate states and findings. We study two network parameters, (1) the parameter governing the assignment of probability values to the network, and (2) the parameter denoting whether the network nodes represent variables with two or more than two values. The experimental results demonstrate that the binary simplification computes diagnoses with similar accuracy to the full multivalued network. We discuss the implications of these parameters, as well other network parameters, for knowledge engineering for medical applications.

Anti-inflammatory activity of resins from some species of the plant family Burseraceae.

The anti-inflammatory activities of extracts from the resins of four species of the plant family Burseraceae, Boswellia dalzielli, Boswellia carteri (gum olibanum), Commiphora mukul, and Commiphora incisa, were studied. The aqueous extracts of the resins of B. dalzielli, C. incisa, and C. mukul significantly inhibited both the maximal edema response and the total edema response during 6 h of carrageenan-induced rat paw edema. The octanordammarane triterpenes, mansumbinone and mansumbinoic acid, isolated from the resin of C. incisa, were separated and tested. Administered prophylactically, mansumbinone proved to be more than 20 times less potent than indomethacin and prednisolone in inhibiting carrageenan-induced rat paw edema. However, the molar potency of mansumbinoic acid was within one order of magnitude of those of indomethacin and prednisolone. The anti-inflammatory action of the acid on the carrageenan-induced edema was dose-related between 1.3 x 10(-5) and 2.5 x 10(-4) mol kg-1 when given before the inflammatory stimulus. The acid was able to reverse an established carrageenan-induced inflammatory response when administered 2 h after induction. Daily administration of mansumbinoic acid at a single dose level (1.5 x 10(-4) mol kg-1) significantly reduced joint swelling in adjuvant arthritis in rats. The results indicated that this compound is worthy of further investigation as an anti-inflammatory drug.

Allelochemicals in foliage of unfavored tree hosts of the gypsy moth,Lymantria dispar L. : 1. Alkaloids and other components ofLiriodendron tulipifera L. (Magnoliaceae),Acer rubrum L. (Aceraceae), andCornus florida L. (Cornaceae).

Early theories on plant chemical defense against herbivory emphasized that apparent and unapparent plants were primarily defended by different types of compounds. More and more evidence suggests that both quantitative and qualitative defenses are found in apparent plants and that they can play a defensive role against herbivores. A survey of the literature on the gypsy moth suggests not only that there is a large variety of qualitative compounds, as well as the expected quantitative ones, but that unfavored hosts of the gypsy moth are associated with the presence of alkaloids. Foliage of three tree species,Liriodendron tulipifera L.,Acer rubrum L., andCornus florida L., was examined to confirm the presence of alkaloids and other major secondary metabolites. The known sesquiterpene lactone, lipiferolide, and the sugar derivative, liriodendritol, were components ofL. tulipifera leaves, along with a bisphenylpropanoid previously found only in nutmeg. Alkaloid content [i.e., (-)-N-methylcrotsparine content] was low and leaves tested positive for HCN. Leaves ofA. rubrum L. were examined for the presence of gramme, but none could be detected. No alkaloids were detected inCornus florida.

Allelochemicals in foliage of unfavored tree hosts of the gypsy moth,Lymantria dispar L. : 2. Seasonal variation of saponins inilex opaca and identification of saponin aglycones.

A greater variety of qualitative chemical defenses has been reported in eastern forest trees than might be expected from current interpretation of the plant apparency theory. For the gypsy moth there is an association between the occurrence of alkaloids and unfavorability of certain tree species, as well as the presence of saponins. The latter association, however, is not statistically significant. Species in the genusIlex have been reported to contain both alkaloids and saponins (Barbosa and Krischick, 1987). In this study, determinations were made of the occurrence of alkaloids and saponins inI. opaca and their changes in concentration over time. No alkaloids were detected. Saponins were isolated, and the aglycone siaresinolic acid was identified. Saponin concentration changes seasonally, being highest in early May and lowest in early June leaves.

Trials with a live attenuated rubella virus vaccine, Cendehill strain.

This report summarizes closed, family, and open studies conducted sequentially over a 10 month period with the Cendehill rubella virus vaccine in more than 16,000 children and adolescents. This strain of rubella was attenuated by serial propagation on primary rabbit kidney cell cultures. Inoculation of the Cendehill vaccine produced seroconversion in 97% of the 3589 susceptible (seronegative) vaccinated persons. There was no spread of the virus to susceptible controls living in close contact with those vaccinated. The vaccine was well tolerated. No arthritis or arthralgia occurred in 860 female subjects 13-18 years of age who were included in the study. The Cendehill vaccine would appear to meet the requirements of an acceptable vaccine.

Trials with a live attenuated rubella virus vaccine, Cendehill strain

This report summarizes closed, family, and open studies conducted sequentially over a 10 month period with the Cendehill rubella virus vaccine in more than 16,000 children and adolescents. This strain of rubella was attenuated by serial propogation on primary rabbit kidney cell cultures. Inoculation of the Cendehill vaccine produced seroconversion in 97 percent of the 3589 susceptible (seronegative) vaccinated persons. There was no spread of the virus to susceptible controls living in close contact with those vaccinated. The vaccine was well tolerated. No arthritis or arthralgia occured in 860 female subjects 13-18 years of age who were included in the study. The Cendehill vaccine would appear to meet the requirements of an acceptable vaccine (Summary)

Clinical trials in Jamaica with the cendehill rubella vaccine - abstract

This paper presents our findings from a closed study involving 54 children, a family study involving 100 families and an open study involving more than 16,000 children, ages five years to 15 years, in an urban area in Jamaica. The vaccine produced good immunogenic response and did not spread the disease to susceptible controls in contact with the vaccinees. Our experience with the Cendehill rubella vaccine has shown that it has all the requirements of an acceptable vaccine. It produced sero-conversion in a large percentage of sero-negative vaccinees; it did not spread the disease to sero negative controls living in close contact; it produced only mild side effects in childern, and there was no evidence of arthralgia or arthritiis in vaccinees(AU)