Gene technology-based antimetabolite design: the use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties.     

Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pK_as of 14 ionizable residues. An all-atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pK_as in the protein, in contrast to multiple simulations for the free energy perturbation method. pK_int shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson-Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long-range interactions on the pK_int shifts is discussed. © 1994 Wiley-Liss, Inc.     

Cultural responsibility in the preservation of local economic plant resources

Understanding the relationships between indigenous people and their threatened economic plants can aid the conservation effort on many levels. Understanding ethnic perceptions of the taxon is critical toin situ andex situ conservation projects and enhances the accompanying educational effort. Examples are discussed from the experience of a grassroots conservation group in southwestern United States, Native Seed/SEARCH. Four levels of economic plant vulnerability are examined among 1) wild-harvested plants, 2) husbanded wild plants, 3) domesticates, and 4) wild relatives of domesticates. Legal interpretations of endangered husbanded and domestic plants are discussed, and further documentation encouraged. Genetic dynamism of threatened indigenous crops is examined and the concept of Systems Conservation (i.e. the plant/human interactive systems) is introduced. Guidelines are offered for incorporating better cultural responsibility intoex situ conservation strategies. The concept of Biocultural Restoration is introduced with an example from an O'odham community. Examples are given of ways indigenous peoples and their knowledge can assist in the conservation effort.     

Interactions between nitrate and ammonium during uptake by carob seedlings and the effect of the form of earlier nitrogen nutrition

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were used in two sets of experiments in order to evaluate; (1) the reciprocal effects of each nitrogen form on net uptake of nitrate and ammonium, and (2) the effect of earlier nitrogen nutrition on ammonium versus nitrate uptake. In the former group of experiments we studied the kinetics of nitrate and ammonium uptake as well as the interference of each of the two forms with net uptake of ammonium and nitrate by both nitrogen depleted and nitrogen fed carob seedlings. On the whole, nitrogen depletion led to increase in both affinity and V_max of the system for both forms of nitrogen, at the same time as the effects of nitrate on uptake of ammonium and vice versa were concentration dependent. In the second group of experiments the effects of earlier nitrogen nutrition on nitrate and ammonium uptake were characterized, and in this case we observed that: (a) if only one form of N was supplied, ammonium was taken up in greater amounts than nitrate; (b) the presence of ammonium enhanced nitrate uptake; (c) ammonium uptake was inhibited by nitrate; (d) there was a significant effect of the earlier nitrogen nutrition on the response of the plants to a different nitrogen source. The latter was evident mainly as regards ammonium uptake by plants grown in ammonium nitrate. The interactions between nitrate and ammonium uptake systems are discussed on the basis of the adaptation to the nitrogen source during early growth.     

Estimating regional carbon stocks and spatially covarying edaphic factors using soil maps at three scales

Most estimates of regional and global soil carbon stocks are based on extrapolations of mean soil C contents for broad categories of soil or vegetation types. Uncertainties exist in both the estimates of mean soil C contents and the area over which each mean should be extrapolated. Geographic information systems now permit spatially referenced estimates of soil C at finer scales of resolution than were previously practical. We compared estimates of total soil C stocks of the state of Maine using three methods: (1) multiplying the area of the state by published means of soil C for temperate forests and for Spodosols; (2) calculating areas of inclusions of soil taxa in the 1:5,000,000 FAO/UNESCO Soils Map of the World and multiplying those areas by selected mean carbon contents; and (3) calculating soil C for each soil series and map unit in the 1:250,000 State Soil Geographic Data Base (STATSGO) and summing these estimates for the entire state. The STATSGO estimate of total soil C was between 23% and 49% higher than the common coarse scale extrapolations, primarily because STATSGO included data on Histosols, which cover less than 5% of the area of the state, but which constitute over one-third of the soil C. Spodosols cover about 65% of the state, but contribute less than 39% of the soil C. Estimates of total soil C in Maine based on the FAO map agreed within 8% of the STATSGO estimate for one possible matching of FAO soil taxa with data on soil C, but another plausible matching overestimated soil C stocks. We also compared estimates from the 1:250,000 STATSGO database and from the 1:20,000 Soil Survey Geographic Data Base (SSURGO) for a 7.5 minute quadrangle within the state. SSURGO indicated 13% less total soil C than did STATSGO, largely because the attribute data on depths of soil horizons in SSURGO are more specific for this locality. Despite localized differences, the STATSGO database offers promise of scaling up county soil survey data to regional scales because it includes attribute data and estimates of areal coverage of C-rich inclusions within map units. The spatially referenced data also permit examination of covariation of soil C stocks with soil properties thought to affect stabilization of soil C. Clay content was a poor predictor of soil C in Maine, but drainage class covaried significantly with soil C across the state.     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Contrasting effects of PACAP and carbachol on [Ca]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca²⁺]_i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca²⁺]_i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca²⁺]_i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca²⁺. Carbachol also increased [Ca²⁺]_i in a biphasic manner, but it mobilized intracellular Ca²⁺ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca²⁺ entry, this being accompanied by a more marked and prolonged elevation of IP₃ and IP₄ than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca²⁺ handling through PACAP receptors than with muscarinic M₁ receptors.     

Oxidative processes as indicators of chemical stress in marine bivalves

Deleterious effects of environmental contaminants could be due to enhanced prooxidant forces overcoming antioxidant defences. Before practical biomarkers based on free radical biology will be generally accepted and validated in situ, additional research is required concerning normal physiological and environmental influences on the relevant systems. The aims of this study were to evaluate in situ the importance of oxyradical production in the presence and absence of pollutants and to characterize some antioxidant systems in Mytilus edulis L. Specimens of M. edulis L. were transplanted from a reference site (Franquelin) to Baie Comeau (Baie des Anglais), on the North shore of the St. Lawrence maritime estuary, where are found aluminium and pulp and paper plants. An oxidative stress was observed in mussels submitted to a chronic exposure in the polluted environment. Variations of pro-and anti-oxidant molecules involved in oxidative processes were related in part to seasonal and physico-chemical influences. Catalase activity, malondialdehyde and glutathione concentrations will be useful as biomarkers of stress in situ since they react to anthropogenic influence and to abiotic factors such as emersion period and temperature.     

小鼠卵激活过程中胞质游离Ca~(2 )的变化及孤雌发育研究

乙醇和电刺激均可使小鼠MⅡ期卵母细胞激活并在体外孤雌发育至囊胚。小鼠卵对乙醇十分敏感。用7%—8%乙醇处理5min后95%以上的卵母细胞(卵龄为HCG注射后18—19h)内形成原核。3—4次电刺激后卵的激活率为71.58%;仅刺激1次卵的激活率为63.63%。乙醇刺激可诱导卵内游离Ca~(2 )浓度出现多次升高;单一电刺激仅能诱导卵内游离Ca~(2 )浓度出现1次升高;多次电刺激可诱导卵内游离Ca~(2 )浓度多次升高,而且电刺激次数与Ca~(2 )浓度升高成一一对应关系。对于电刺激,介质中足够量的Ca~(2 )对卵激活至关重要。在无Ca~(2 )的介质中,电刺激很难使卵激活。正常受精刺激诱导卵内游离Ca~(2 )浓度出现多次有规律的升高。实验结果表明,卵母细胞激活过程中胞质游离Ca~(2 )浓度重复多次升高可促使卵母细胞恢复成熟分裂。     

烧伤狗浆血游离氨基酸的动态变化

烧伤狗浆血游离氨基酸的动态变化黎君友,周幼勤,赖业馥,赵有,伊少杰ＳｅｑｕｅｎｔｉａｌＣｈａｎｇｅｓｉｎＰｌａｓｍａＦｒｅｅＡｍｉｎｏＡｃｉｄＣｏｎｃｅｎｔｒａｔｉｏｎｉｎＢｕｒｎｅｄＤｏｇｓ￥Ｌｉｊｕｎｙｏｎ;Ｚｈｏｕｙｏｕｑｉｎ;Ｌａｉｙｅｆｕ;...