Interaction of isoquinoline alkaloid palmatine with deoxyribonucleic acids: binding heterogeneity, and conformational and thermodynamic aspects

The binding heterogeneity, conformational aspects, and energetics of the interaction of the cytotoxic plant alkaloid palmatine have been studied with various natural and synthetic DNAs. The alkaloid binds to calf thymus and Escherichia coli DNA that have mixed AT and GC sequences in almost equal proportions with positive cooperativity, while, with Clostridium perfringens and Micrococcus lysodeikticus DNA with predominantly high AT and GC sequences, respectively, noncooperative binding was observed. On further investigation with synthetic DNAs, the binding was observed to be cooperative with polymers like poly(dA).poly(dT) and poly(dG).poly(dC) having poly(purine)poly(pyrimidine) sequences, while with polymers poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT) and poly(dG-dC).poly(dG-dC), which have alternating purine-pyrimidine sequences, a non-cooperative binding phenomenon was observed. This suggests the binding heterogeneity of palmatine to the two types of sequences of base pairs. Circular dichroism (CD) studies revealed that the binding induced conformational changes in all the DNAs, but more importantly, the bound alkaloid molecules acquired induced optical activity, and the extent was dependent on the AT content and showed AT base-pair specificity. Energetics of the interaction of the alkaloid studied by highly sensitive isothermal titration calorimetry revealed that the binding was in most cases exothermic and favored by both enthalpy and entropy changes, while, in the case of the homo and hetero AT polymers, the same was predominantly entropy-driven. This study defines base-pair-dependent heterogeneity, conformational aspects, and energetics of palmatine binding to DNA.     

Relative binding affinities of chlorophylls in peridinin–chlorophyll–protein reconstituted with heterochlorophyllous mixtures

Peridinin–chlorophyll–protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these “half-mers”, homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.     

The influence of leaf size and shape on leaf thermal dynamics: does theory hold up under natural conditions?

Laboratory studies on artificial leaves suggest that leaf thermal dynamics are strongly influenced by the two‐dimensional size and shape of leaves and associated boundary layer thickness. Hot environments are therefore said to favour selection for small, narrow or dissected leaves. Empirical evidence from real leaves under field conditions is scant and traditionally based on point measurements that do not capture spatial variation in heat load. We used thermal imagery under field conditions to measure the leaf thermal time constant (τ) in summer and the leaf‐to‐air temperature difference (?T) and temperature range across laminae (T_range) during winter, autumn and summer for 68 Proteaceae species. We investigated the influence of leaf area and margin complexity relative to effective leaf width (w_e), the latter being a more direct indicator of boundary layer thickness. Normalized difference of margin complexity had no or weak effects on thermal dynamics, but w_e strongly predicted τ and ?T, whereas leaf area influenced T_range. Unlike artificial leaves, however, spatial temperature distribution in large leaves appeared to be governed largely by structural variation. Therefore, we agree that small size, specifically w_e, has adaptive value in hot environments but not with the idea that thermal regulation is the primary evolutionary driver of leaf dissection.     

Kinetic characteristics of L-lysine α- oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D: Substrate specificity and allosteric effects

The present work aims to investigate the kinetic characteristics of homodimer enzyme L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D, taking into account allosteric effects. The enzyme was first shown to reveal positive cooperativeness, h=2.05±0.15. Using additional opportunities of Hill coefficient the value of the Michaelis–Menten constant has been estimated, K_m=1.015?10^?5М, indicating high strength of substrate binding to the active site of each subunit. High selectivity and absolute L-stereospecificity of the enzyme were shown. The inhibition of L-lysine conversion by non-cleavable lysine analogs as well as the reaction product was found out to take place. These effects have been evaluated only as the inhibition coefficients (%). A more detailed study of these inhibition effects was complicated because of the cooperativeness of enzyme subunits mentioned above. The kinetic scheme of L-lysine α-oxidase was proposed involving parallel-subsequent action of each of two subunits in the catalytic act.We think that the results obtained will be useful for studying the kinetic properties of other multi-subunit enzymes and improve understanding of the mechanisms of their action.     

Aging does not compromise in vitro oscillation of the suprachiasmatic nuclei but makes it more vulnerable to constant light

Circadian regulation of behavior worsens with age, however, the mechanism behind this phenomenon is still poorly understood. Specifically, it is not clear to what extend the ability of the circadian clock in the suprachiasmatic nuclei (SCN) to generate the rhythm is affected by aging. This study aimed to ascertain the effect of aging on the functioning of the SCN of mPer2^Luciferase mice under unnatural lighting conditions, such as constant light (LL). Under LL, which worsened the age-induced effect on behavioral rhythms, a marginal age-dependent effect on in vitro rhythmicity in explants containing the middle, but not the rostral/caudal, regions of the SCN was apparent; the proportion of mice in which middle-region SCN explants were completely arrhythmic or had an extremely long period (>30 h) was 47% in aged mice and 27% in adults. The results suggest that in some of the aged animals, LL may weaken the coupling among oscillators in specific sub-regions of the SCN, leaving other sub-regions better synchronized. In the standard light/dark cycle and in constant darkness, the SCN ability to produce bioluminescence rhythms in vitro was not compromised in aged mice although aging significantly affected their SCN-driven locomotor activity rhythms. Therefore, our results demonstrate that although age worsened the SCN output rhythm, the SCN molecular core clock mechanism itself was relatively resilient to aging in these same animals. The results suggest the involvement of pathways downstream of the core clock mechanism which are responsible for this phenomenon.     

A comparative study of warheads for design of cysteine protease inhibitors

The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude.     

Influence of the cellular environment on ligand binding kinetics at membrane-bound targets

While historically ‘in vitro’ binding data were obtained by analyzing equilibrium experiments, kinetic data are increasingly appreciated to provide information on the time a particular compound remains bound to its target. This information is of biological importance to understand the molecular mechanism of a drug not only to evaluate the time a particular receptor/enzyme is blocked in the case of antagonists/inhibitors but also to investigate its contribution to the efficacy to mediate signaling in the case of agonists. There is accumulating evidence that many drugs binding to either membrane-bound receptors or enzymes are found to display long duration of action which can be ascribed to slow dissociation from their target proteins. In the present review three such examples are discussed which encompass ligands that bind to membrane-bound proteins and from which it appears that the tight binding kinetics is influenced by the cellular/membrane environment of the target protein.     

Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.     

Melatonin is Involved in Regulation of the Expression of Neural Cell Adhesion Molecules in the Rat Brain

Cell adhesion molecules play a diverse role in neural development, signal transduction, structural linkage to extracellular and intracellular proteins, synaptic stabilization, neurogenesis, and learning. Neural cell adhesion molecules (NCAM) are members of the immunoglobulin superfamily and are involved in synaptic rearrangements in the mature brain. There are three major NCAM isoforms: NCAM 180, NCAM 140, and NCAM 120. Several studies reported that NCAM play a central role in memory formation. We investigated the effects of melatonin on the expression of NCAM in the hippocampus, cortex, and cerebellum of rats. The levels of NCAM isoforms were determined by Western blotting. After administration of melatonin for 7 days, the expression of NCAM 180 increased both in the hippocampus and in the cortex, as compared with the control. In contrast, in rats exposed to constant illumination for 7 days (a procedure that inhibits endogenous production of melatonin), levels of NCAM 180 dropped in the hippocampus and became undetectable in the cortex and cerebellum. Levels of NCAM 140 in the hippocampus of light-exposed rats also decreased. There was no change in the expression of NCAM 120 in any brain region. This is the first report indicating that melatonin exerts a modulatory effect on the expression of NCAM in brain areas related to realization of cognitive functions. Melatonin may be involved in structural remodeling of synaptic connections during memory and learning processes.     

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus-Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus-Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus-Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 x 10(-9) M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of 相似文献