2,4,5-Tris(alkoxyaryl)imidazoline derivatives as potent scaffold for novel p53-MDM2 interaction inhibitors: Design,synthesis, and biological evaluation

Imidazoline-based small molecule inhibitors of p53-MDM2 interaction intended for the treatment of p53 wild-type tumors are the promising structures for design of anticancer drugs. Based on fragment approach we have investigated a key role of substituents in cis-imidazoline core for biological activity of nutlin family compounds. Although the necessity of the substituents in the phenyl rings of cis-imidazoline has been shown, there are no studies in which the replacements of a halogen by other substituents have been investigated. A series of simple cis-imidazoline derivatives containing halogen, hydroxy and alkoxy-substituents were synthesized. The biological activity of the compounds was studied using assays of cytotoxicity (MTT) and p53 level. It was found that the hydroxyl-derivatives were not cytotoxic whereas the alkoxy analogues were the same or more active as halogen-substituted compounds in cell viability test. The synthesized alkoxy derivatives induced an increase of p53 level and did not promote necrotic cell death in the concentration up to 40 µM.     

Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb (<Emphasis Type="Italic">Rheum rhabarbarum</Emphasis> L.)

Experiments were conducted to elucidate the hormonal induction and regulation of rhizome growth in rhubarb (Rheum rhabarbarum L.). It was found that ethylene is the key regulator of rhizome induction and development. The role of jasmonic acid (JA) and its interaction with ethylene in rhizome induction and growth were also examined. Both ethylene and JA have a significant effect on promoting rhizome formation in vitro. Conversely, the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) (1.5 μM) inhibited rhizome induction in multiple-shoot clumps in vitro, and suppressed the stimulatory effects of exogenously applied ethephon (1 mg l⁻¹) and JA (10 ng l⁻¹) in promoting mini-rhizome formation, further confirming the role of endogenous ethylene in the process. In addition, rhizome growth was significantly enhanced in the presence of both ethylene and JA (ethephon 1 mg l⁻¹ and JA 10 ng l⁻¹) compared to JA alone. These results suggest that endogenous ethylene is the key regulator of rhizome growth in rhubarb and JA promotes rhizome formation, possibly through inducing endogenous ethylene synthesis.     

Integrity of gut mucosa during anaesthesia in major abdominal surgery

The aim of the study was to examine a perfusion and integrity of small bowel in 60 subsequent patients during the major open abdominal surgery which lasted from 2 to 7 hours. Two samples of the intestinal mucosa were removed: at the beginning, and at the end of the surgical procedure in general anaesthesia. A mucosal injury was classified into 4 grades. pH, PCO2 and lactate level were measured in the blood samples from the arterial and mesenteric vein in one hour time intervals. The changes of intestinal mucosa were found in 31 patients (51.7%): in 19 patients (31.7%) grade 1 changes were recorded, in 10 patients (16.7%) grade 2, and in 2 patients (3.3%) grade 3. Grade 4 lesions were not recorded. There was a statistically significant correlation between grades of the mucosal damage and the surgery duration (p = 0.001). Analysis during the one hour intervals showed that there was no exact time point when the significant aggravation of the pathohistological changes in intestinal mucosa occurred. However, when patients were allocated into two subgroups with surgical procedures lasting less than 4 hours and more than 4 hours, there was a statistically significant difference in the grades of mucosal damage between subgroups (p < 0.05). More biopsies without pathohistological changes were observed in the patients whose procedure duration was < 4 hours. A significantly higher lactate concentrations in arterial and mesenteric venous blood were observed in the patients with pathohistological changes at 6 hours time point as compared to 2 hour time point in the patients without pathohistological changes (p < 0.05). During the open abdominal surgery in general anaesthesia, the length of the procedure influences the grade of the intestinal mucosa injury. Deterioration of the pathohistological findings in the intestinal mucosa correlates with high lactate blood level, suggesting that the cause of these changes may result from tissue hypoxia.     

Dual mode of glucagon receptor internalization: role of PKCα, GRKs and β-arrestins

Glucagon levels are elevated in diabetes and some liver diseases. Increased glucagon secretion leads to abnormal stimulation of glucagon receptors (GRs) and consequent elevated glucose production in the liver. Blocking glucagon receptor signaling has been proposed as a potential treatment option for diabetes and other conditions associated with hyperglycemia. Elucidating mechanisms of GR desensitization and downregulation may help identify new drug targets besides GR itself. The present study explores the mechanisms of GR internalization and the role of PKCα, GPCR kinases (GRKs) and β-arrestins therein. We have reported previously that PKCα mediates GR phosphorylation and desensitization. While the PKC agonist, PMA, did not affect GR internalization when tested alone, it increased glucagon-mediated GR internalization by 25–40% in GR-expressing HEK-293 cells (HEK-GR cells). In both primary hepatocytes and HEK-GR cells, glucagon treatment recruited PKCα to the plasma membrane where it colocalized with GR. We also observed that overexpression of GRK2, GRK3, or GRK5 enhanced GR internalization. In addition, we found that GR utilizes both clathrin- and caveolin-mediated endocytosis in HEK-GR cells. Glucagon triggered translocation of both β-arrestin1 and β-arrestin2 from the cytosol to the perimembrane region, and overexpression of β-arrestin1 and β-arrestin2 increased GR internalization. Furthermore, both β-arrestin1 and β-arrestin2 colocalized with GR and with Cav-1, suggesting the possible involvement of these arrestins in GR internalization.     

Mutator effects and mutation signatures of editing deaminases produced in bacteria and yeast

Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenosine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This cannot be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation.     

Systemic redox imbalance in stable chronic obstructive pulmonary disease

Context: Increased oxidative burden is found in chronic obstructive pulmonary disease (COPD).

Objective: To assess the association of ceruloplasmin, albumin, bilirubin, transferrin, thiols and malondialdehyde (MDA) with stable COPD.

Materials and methods: Oxidative stress markers measured in 106 COPD patients and 45 healthy subjects were evaluated.

Results: Higher ceruloplasmin and MDA, and lower albumin, transferrin and thiols in COPD patients were found. Ceruloplasmin was the strongest single predictor of COPD. The model combining ceruloplasmin, albumin and thiols improved their individual diagnostic performances.

Conclusions: Diagnostic characteristics of ceruloplasmin, albumin, transferrin, thiols and MDA suggest their potential value as additional tools in disease diagnosis.     

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.     

Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan

Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1-->4)-beta-mannan/galacto-(1-->4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.