MNDO study of the mechanism of the inhibition of cysteine proteinases by diazomethyl ketones

Diazomethyl ketones are one of the most effective irreversible inhibitors of cysteine proteinases and are therefore very important in drug design. In the present study a mechanism of inactivation is proposed based on the results of model MNDO calculations of the possible pathways. It was found that the mercaptide nucleophile, on approaching the carbonyl carbon as in the catalytic reaction path, binds to the inner diazo nitrogen. The intermediate thus formed can rearrange giving a stable product, -thioketone, and molecular nitrogen, with a considerable energy gain. The energy barrier to this process is equal to 36.9 kcal/mol, and corresponds to a pyramidal transition state with the vertex at the methylene carbon and the base formed by the carbonyl, thiol, and diazo groups. The energy barrier can be lowered on deprotonation of the intermediate. Based on the results obtained it was concluded that good irreversible inhibitors of cysteine proteases must fulfil two structural requirements: i) the dimensions and charge distribution must be similar to those of the peptide bond and ii) a second electrophilic center must be present in the neighbourhood of the carbonyl carbon. These are requirements which are satisfied by other strong cysteine proteinase inhibitors: -chloroketones and -ketooxiranes.     

The Immunomodulatory Activity of Peptides Related to the DNA Contacting Loop of p53 Protein

Taking into account the sequence homology existing between thymopoietin II and the DNA-binding domain of p53 protein, a series of octapeptides was synthesized, related to the wild p53 type protein as well as to its mutated forms, appearing in some human tumours. The wild type octapeptide has immunostimulative activity with regard to the humoral immune response, but is inactive in the cellular immune response. The mutated peptides of p53 differ in their immunomodulatory activity from the wild type octapeptide. The Ser5 analogue of the wild type peptide is a strong stimulant of the humoral immune response and enhances TNF-α production, while at the same time suppressing the cellular immune response. The data suggest that the mutations of p53, which favour tumour development and growth, may also change the immune activity of respective p53 fragments.     

Conformations of the d-glucarolactones and d-glucaric acid in solution

The conformations of d-glucaric acid (1), d-glucaro-1,4-lactone (2), d-glucaro-6,3-lactone (3), and d-glucaro-1,4:6,3-dilactone (4) in solution were investigated by ¹H-n.m.r. and ¹³C-p.F.t., n.m.r. spectroscopy. The solvents used were deuterium oxide, methanol-d₄, and dimethyl sulfoxide-d₆, and praseodymium chloride was employed as a lanthanide shift-reagent. For 2, it was found that the conformational equilibrium ³E(d)

E₃(d) exists in solution, and that the OH-5 group tends to occupy the position over the lactone ring in the favored E₃(d),gg conformation. The n.m.r. data for 3 indicated that the conformational equilibrium is shifted in favor of the ⁴E(d)

E₄(d),gt conformation in solution. The dienvelope conformation ³E:E₄(d) was found to be the favored conformation of 4. For 1, a conformational equilibrium between one planar, zigzag form and two sickle forms was indicated by the n.m.r. data observed. ¹³C-N.m.r. spectroscopy proved to be a convenient method for monitoring the lactonization of 1, and the hydrolysis of its lactones. Lactones other than 2–4 were not found in solutions prepared from 1–4, either during their mutarotation or after equilibration at 30°.     

Increase in chloroplastic thiol groups by SO2 and its effect on light modulation of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase

In broken spinach chloroplasts the total amount of thiol groups is about 3.7 mol mg^-1 chlorophyll. Two thirds are represented by the masked form (which is only titratable after unfolding of the protein). Of the free groups, those reacting with NBD·Cl (1.2–2.0 mol mg^-1 chlorophyll) seem to be undergoing oxidation more readily than those reacting with DTNB (1.0 mol mg^-1 chlorophyll). SO₂ application causes a maximal increase of 25% in free thiols, and doubles the amount of the masked thiols. The light triggered increase in SH, which starts at an elevated level, runs parallel to that of the controls. SO₂ application of 1.8 mg m^-3 (=28 nmol l^-1) for 1 h does not affect the dark level of NADP-GPD but enhances the light modulation by increasing the ratio of activation. This enhancement is explained by an increase in masked thiol groups during the preceding fumigation period.Abbreviations DTNB 5,5 dithiobis-2-nitrobenzene-2-oxa-1,3 diazole - NBD·Cl 7-chloro-4-nitrobenzene-2-oxa-1,3 diazole - PCMB p-chloromercuribenzoate - SDS sodium dodecylsulfate - NADP-GPD NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2[N-Morpholino]ethanesulfonic acid - PGA 3-phosphoglyceric acid     

Changing the way we think about global change research: scaling up in experimental ecosystem science

Scaling is a naturally iterative and bi‐directional component of problem solving in ecology and in climate science. Ecosystems and climate systems are unquestionably the sum of all their parts, to the smallest imaginable scale, in genomic processes or in the laws of fluid dynamics. However, in the process of scaling‐up, for practical purposes thewhole usually has to be construed as a good deal less than this. This essay demonstrates how controlled large‐scale experiments can be used to deduce key mechanisms and thereby reduce much of the detail needed for the process of scaling‐up. Collection of the relevant experimental evidence depends on controlling the environment and complexity of experiments, and on applications of technologies that report on, and integrate, small‐scale processes. As the role of biological feedbacks in the behavior of climate systems is better appreciated, so the need grows for experimentally based understanding of ecosystem processes. We argue that we cannot continue as we are doing, simply observing the progress of the greenhouse gas‐driven experiment in global change, and modeling its future outcomes. We have to change the way we think about climate system and ecosystem science, and in the process move to experimental modes at larger scales than previously thought achievable.     

Trichinella spiralis infection affects p47(phox) protein expression in guinea-pig alveolar macrophages

To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.     

Effect of l-carnitine on liver cell membranes in ethanol-intoxicated rats

Ethanol intoxication is characterized by changes in cell metabolism which alter the structure and function of cell membrane components, including phospholipids and integral membrane proteins. The interaction of food nutrients with ethanol may modulate alcohol toxicity. One such compound is l-carnitine (l-3-hydroxy-4-N,N,N-trimethylaminobutyrate), which is also an antioxidant. Here we investigate l-carnitine as an antioxidant and assess its effect on the composition and electrical charge of liver cell membranes in ethanol-intoxicated rats. Qualitative and quantitative phospholipid composition and the presence of integral membrane proteins were determined by high performance liquid chromatography (HPLC). Electrophoresis was used to determine the surface charge density of the rat liver cell membranes. Ethanol increased phospholipid levels and altered the level of integral proteins as determined by decreased phenylalanine (Phe), cysteine (Cys) and lysine (Lys). Ethanol significantly enhanced changes in the surface charge density of the liver cell membranes. l-Carnitine administration to ethanol-intoxicated rats significantly protects phospholipids and proteins against oxidative modifications. Therefore, the beneficial effect of l-carnitine may be connected to its ability to scavenge free radicals.