Markers of cellular senescence in zero hour biopsies predict outcome in renal transplantation

Although chronological donor age is the most potent predictor of long-term outcome after renal transplantation, it does not incorporate individual differences of the aging-process itself. We therefore hypothesized that an estimate of biological organ age as derived from markers of cellular senescence in zero hour biopsies would be of higher predictive value. Telomere length and mRNA expression levels of the cell cycle inhibitors CDKN2A (p16INK4a) and CDKN1A (p21WAF1) were assessed in pre-implantation biopsies of 54 patients and the association of these and various other clinical parameters with serum creatinine after 1 year was determined. In a linear regression analysis, CDKN2A turned out to be the best single predictor followed by donor age and telomere length. A multiple linear regression analysis revealed that the combination of CDKN2A values and donor age yielded even higher predictive values for serum creatinine 1 year after transplantation. We conclude that the molecular aging marker CDKN2A in combination with chronological donor age predict renal allograft function after 1 year significantly better than chronological donor age alone.     

Anticonvulsant activity of some xanthone derivatives

A series of appropriate alkanolamine and amide derivatives of xanthone were prepared and evaluated for anticonvulsant activity using maximal electroshock (MES) and subcutaneous pentylenetetrazole (scMet) induced seizures, and for neurotoxicity (TOX) using the rotorod test on mice and rats. The most promising compounds seem to be the appropriate aminoalkanolic derivatives of 6-chloroxanthone, among which the R-(-) and S-(+)-2amino-1-propanol derivatives of 6-chloro-2-methylxanthone (2(a) and 2(b)) displayed anti-MES activity (in mice) with a protective index (TD(50)/ED(50)) of 6.23<6.85, corresponding to that of phenytoin, carbamazepine and valproate. The most active compound, 2(b), was determined to have an affinity to the benzodiazepine (BDZ) receptor and voltage-dependent Ca(2+) channel (VDCC) by using radioligand binding assays. The enantiomeric purities of 2(a) and 2(b) were determined using an analytical liquid chromatography-mass spectrometry method.     

Transport of micro-opioid receptor agonists and antagonist peptides across Caco-2 monolayer

Milk is the source of β-casomorphins – biologically active peptides with opioid activity – which are suspected to play various roles in the human body. The local influence of exogenous opioid peptides on gastrointestinal functions has been widely reported. After passing the gut barrier, β-casomorphins may affect the functions of immunological system, as well as dopaminergic, serotoninergic and GABA-ergic systems in brain, regulate the opioid receptor development and elicit behavioral effects. However, possibilities and mechanisms of the intestinal transport of β-casomorphins in human body in vivo have not been reported so far. In our research, the transepithelial transport of μ-opioid receptor agonists – human β-casomorphin-5 and 7(BCM5, BCM7) and antagonist – lactoferroxin A (LCF A) have been investigated using Caco-2 monolayer. In order to determine the pathway of investigated peptide transport across Caco-2 monolayer, two directions of the transport (apical to basolateral and basolateral to apical) have been studied. All investigated peptides were transported across the human intestinal cell line Caco-2 and the curves of cumulative amount of transported peptides in time were linear in each case. In addition, the hydrolysis of β-casomorphins during 60 min of experiment by dipeptidyl peptidase IV was observed. The data suggest the possibility of transport of opioid peptides derived from food across human intestinal mucosa.     

Kinase selectivity profiling by inhibitor affinity chromatography

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas. In this case, inhibitors targeting the ATP pocket may cross react with different kinases, as well as with other proteins that bind ATP. This review critically discusses the available approaches for kinase selectivity profiling. It also reviews some examples of inhibitor affinity chromatography applied to inhibitors of kinases and other protein families as a tool to identify and characterize their intracellular targets.     

Modern analytical ultracentrifugation in protein science: look forward, not back

The reasons for replacing the classical strictly two-component velocity and equilibrium analytical ultracentrifugation Svedberg equations by multicomponent equations, applicable in the study of biological macromolecular systems, are given.     

Chronic liver and renal diseases differently affect structure of human serum albumin

Human serum albumin (HSA) binding with endogenous metabolites and drugs is substantially decreased in chronic renal and liver diseases. To test the hypothesis that the decreased binding ability is caused by conformational changes of the protein, we analyzed infrared and Raman spectra of HSA isolated from healthy donors and patients with chronic uremia and liver cirrhosis. Uremia did not affect the secondary structure of HSA but modified the environment of its Asp/Glu residues. Liver cirrhosis increased the amount of extended and beta-structures, modified the environment of Asp/Glu and Tyr side chains, and changed the configuration of disulfide bridges in albumin molecules. The conformational changes of "cirrhotic" albumin were not caused by reversibly bound ligands and resembled a partial unfolding of the protein induced by adsorption on the charcoal surface. The dramatic structural alterations of HSA in liver cirrhosis may be caused by its oxidative modification and might underlie the decreased binding ability and changed body distribution of albumin.     

The effect of catechol O-methylation on radical scavenging characteristics of quercetin and luteolin--a mechanistic insight

The biological effect of flavonoids can be modulated in  vivo due to metabolism. The O-methylation of the catechol group in the molecule by catechol O-methyl transferase is one of the important metabolic pathways of flavonoids. In the present study, the consequences of catechol O-methylation for the pH-dependent radical scavenging properties of quercetin and luteolin were characterized both experimentally and theoretically. Comparison of the pKa values to the pH-dependent TEAC profiles reveals that O-methylation not only affects the TEAC as such but also modulates the effect of changing pH on this radical scavenging activity due to an effect on the pKa for deprotonation. The pH-dependent TEAC curves and computer calculated electronic parameters: bond dissociation energy (BDE) and ionisation potential (IP) even indicate that O-methylation of the luteolin catechol group affects the radical scavenging potential only because it shifts the pKa for deprotonation. O-Methylation of the quercetin catechol moiety affects radical scavenging capacity by both an effect on the pKa, and also by an effect on the electron and hydrogen atom donating properties of the neutral (N) and the anionic (A) form of the molecule. Moreover, O-methylation of a catechol OH-group in quercetin and luteolin has a similar effect on their TEAC profiles and on calculated parameters as replacement of the OH-group by a hydrogen atom. Altogether, the results presented provide new mechanistic insight in the effect of catechol O-methylation on the radical scavenging characteristics of quercetin and luteolin.     

Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, we examined conditional null alleles of both the Sonic hedgehog and Smoothened genes. While the removal of Shh signaling in these animals resulted in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus was dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals formed significantly fewer neurospheres. The loss of hedgehog signaling also resulted in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon.     

Extent of metal ion-sulfur binding in complexes of thiouracil nucleosides and nucleotides in aqueous solution

Previously published stability constants of several metal ion (M2+) complexes formed with thiouridines and their 5'-monophosphates, together with recently obtained log K(M(U))(M) versus pK(U)(H) plots for M2+ complexes of uridinate derivatives (U-) allowed now a quantitative evaluation of the effect that the exchange of a (C)O by a (C)S group has on the stability of the corresponding complexes. For example, the stability of the Ni2+, Cu2+ and Cd2+ complexes of 2-thiouridinate is increased by about 1.6, 2.3, and 1.3 log units, respectively, by the indicated exchange of groups. Similar results were obtained for other thiouridinates, including 4-thiouridinate. The structure of these complexes and the types of chelates formed (involving (N3)- and (C)S) are discussed. A recently advanced method for the quantification of the chelate effect allows now also an evaluation of several complexes of thiouridinate 5'-monophosphates. In most instances the thiouracilate coordination dominates the systems, allowing only the formation of small amounts of phosphate-bound isomers. Among the complexes studied only the one formed by Cu2+ with 2-thiouridinate 5'-monophosphate leads to significant amounts of the macrochelated isomer, which means that in this case Cu2+ is able to force the nucleotide from the anti to the syn conformation, allowing thus metal ion binding to both potential sites and this results in the formation of about 58% of the macrochelated isomer. The remaining 42% are species in which Cu2+ is overwhelmingly coordinated to the thiouracilate residue; Cu2+ binding to the phosphate group occurs in this case only in trace amounts.     

Only an early nitric oxide burst and the following wave of secondary nitric oxide generation enhanced effective defence responses of pelargonium to a necrotrophic pathogen

Participation of nitric oxide (NO) in cross-talk between ivy pelargonium (Pelargonium peltatum) leaves and Botrytis cinerea was investigated using electrochemical and biochemical approaches. In response to the necrotroph, leaves initiated a near-immediate NO burst, but the specificity of its generation was dependent on the genetic makeup of the host plant. In the resistant cultivar, a strong NO burst was followed by a wave of secondary NO generation, shown by bio-imaging with DAF-2DA. The epicentre of NO synthesis was located in targeted cells, which exhibited a TUNEL-positive reaction. Soon after the challenge, an elevated concentration of hydrogen peroxide (H(2)O(2)) was correlated with a reversible inhibition of catalase (CAT), ascorbate peroxidase (APX), and suppression of ethylene synthesis. The induced NO generation initially expanded and then gradually disappeared on successive days, provoking noncell-death-associated resistance with an enhanced pool of antioxidants, which finally favoured the maintenance of homeostasis of surrounding cells. By contrast, in the susceptible pelargonium, a weak NO burst was recorded and further NO generation increased only as the disease progressed, which was accompanied by very intensive H(2)O(2) and ethylene synthesis. The pathogen colonizing susceptible cells also acquired the ability to produce considerable amounts of NO and enhanced nitrosative and oxidative stress in host tissues.