The effect of new non-cross resistant antitumour agents on the energy state of human erythrocytes

Multidrug resistance (MDR) of tumour cells is related to the overexpression of ATP-dependent pumps responsible for the active efflux of antitumour agents out of resistant cells. Benzoperimidine and anthrapyridone compounds exhibit comparable cytotoxic activity against sensitive and MDR tumour cells. They diffuse extremely rapidly across the plasma membrane and render the ATP-dependent efflux inefficient. Such uptake could disturb an energy metabolism of normal cells possessing an elevated level of ATP-dependent proteins, especially erythrocytes having a high level of the MRP1, MRP4 and MRP5 proteins. In this study the effect of five antitumour agents: benzoperimidine (BP1), anthrapyridones (CO1, CO7) and reference drugs used in the clinic: doxorubicin (DOX) and pirarubicin (PIRA), on the energetic state in human erythrocytes has been examined. These compounds have various types of structure and kinetics of cellular uptake (slow--DOX, CO7, moderate--PIRA, fast--BP1, CO1) resulting in their different ability to saturate ATP-dependent transporters. The energetic state of erythrocytes was examined by determination of purine nucleotide contents (ATP, ADP, AMP), NAD(+) and values of adenylate energy charge (AEC) using an HPLC method. It was found that the level of nucleotides as well as the AEC value of erythrocytes were not changed during 24 h of incubation with these agents independently of their structure and ability to saturate ATP-dependent pumps. This is a very promising result in view of their potential use in the clinic as antitumour drugs against multidrug resistant cancers.     

Detection and analysis of tumor fluorescence using a two-photon optical fiber probe

The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing as little as 0.3% GFP-expressing cells. TPOFF results were similar to both flow-cytometric analysis of tumor cells after isolation and suspension, and fluorescence determined by microscope images of cryosectioned tumors. TPOFF was then used to measure GFP fluorescence from tumors in live mice. The fiber probe detected fluorescently-labeled Herceptin antibody targeted to HER2-expressing tumors in severe combined immunodeficient mice. Dendrimer nanoparticles targeted by folic acid and having 6-TAMRA as a fluorescent probe were also used to label KB cell tumors in vivo. The fiber probe documented a fourfold increase in tumor fluorescence in animals that received the targeted dendrimer. These results suggest TPOFF can be used as a minimally invasive system for identifying tumor markers and monitoring drug therapy.     

The development of a continuous isothermal titration calorimetric method for equilibrium studies

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12-20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60-180 min to generate 20-30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba(2+) to 18-crown-6 and the binding of cytidine 2'-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.     

Rhizochaete, a new genus of phanerochaetoid fungi

A new basidiomycete genus, Rhizochaete (Phanerochaetaceae, Polyporales), is described. Rhizochaete is characterized by a smooth to tuberculate, pellicular hymenophore and hyphal cords that turn red or violet in potassium hydroxide, monomitic hyphal system of simple or nodose septate hyphae, cystidia, and small, cylindrical to subglobose basidiospores. It morphologically is most similar to Phanerochaete. Analyses of nuclear ribosomal and internal-transcribed spacer region sequence data support a close relationship between Rhizochaete and Phanerochaete. The new taxon R. brunnea, from southern Argentina, is described and illustrated. In addition, the new combinations R. americana, R. borneensis, R. filamentosa, R. fouquieriae and R. radicata are proposed. A key to the species of Rhizochaete is provided.     

Effect of early thermal experience on pituitary-gonadal axis in female rats

The reproductive system fully develops during postnatal stages of life, and as such it may be susceptible to environmental cues such as high temperature. Thus, the purpose of the study was to compare how exposure to 34 degrees Celsius ambient temperature affects pituitary-gonadal axis of immature and adult female rats. Three groups of females at proestrous or metaestrous (n=38) were used in the study. The females were housed in ambient temperature of 34 degrees Celsius (WR group, n=10) or room temperature (CR group, n=16) from birth to adulthood. The females from the third group were acclimated to 34 degrees Celsius as adults (WA group, n=12). In the WR group the onset of puberty was delayed in comparison to the CR group. The plasma PRL level was lower during proestrous and higher during metaestrous in the WR group compared to the CR group. During metaestrous, lower FSH and higher progesterone (P(4)) plasma concentrations were found in the WR females. No changes in LH and oestradiol (E(2)) plasma concentrations were demonstrated. Higher in vitro E(2) and lower P(4) secretions under FSH stimulation were observed in the WR follicles compared to those of the CR group. The WR group also demonstrated higher basal and LH-stimulated luteal in vitro secretion of P(4) than controls. Plasma LH and FSH concentrations during metaestrous were higher in WA females than in the WR group, but PRL level was lower. Follicles of the WA group were unresponsive to FSH with respect to steroid secretion. In addition, LH stimulated luteal E(2) secretion in this group. P(4) release by luteal cells was lower in the WA than in the WR group. We concluded that WR females differ from WA in reproductive system adjustments to rearing temperature and that early thermal experience is more effective in antagonizing the effect of high temperature than acclimation of adult females.     

Early Innate Recognition of Herpes Simplex Virus in Human Primary Macrophages Is Mediated via the MDA5/MAVS-Dependent and MDA5/MAVS/RNA Polymerase III-Independent Pathways

Innate recognition of viruses is mediated by pattern recognition receptors (PRRs) triggering expression of antiviral interferons (IFNs) and proinflammatory cytokines. In mice, Toll-like receptor 2 (TLR2) and TLR9 as well as intracellular nucleotide-sensing pathways have been shown to recognize herpes simplex virus (HSV). Here, we describe how human primary macrophages recognize early HSV infection via intracellular pathways. A number of inflammatory cytokines, IFNs, and IFN-stimulated genes were upregulated after HSV infection. We show that early recognition of HSV and induction of IFNs and inflammatory cytokines are independent of TLR2 and TLR9, since inhibition of TLR2 using TLR2 neutralizing antibodies did not affect virus-induced responses and the macrophages were unresponsive to TLR9 stimulation. Instead, HSV recognition involves intracellular recognition systems, since induction of tumor necrosis factor alpha (TNF-α) and IFNs was dependent on virus entry and replication. Importantly, expression of IFNs was strongly inhibited by small interfering RNA (siRNA) knockdown of MAVS, but this MAVS-dependent IFN induction occurred independently of the recently discovered polymerase III (Pol III)/RIG-I DNA sensing system. In contrast, induction of TNF-α was largely independent of MAVS, suggesting that induction of inflammatory cytokines during HSV infection proceeds via a novel pathway. Transfection with ODN2006, a broad inhibitor of intracellular nucleotide recognition, revealed that nucleotide-sensing systems are employed to induce both IFNs and TNF-α. Finally, using siRNA knockdown, we found that MDA5, but not RIG-I, was the primary mediator of HSV recognition. Thus, innate recognition of HSV by human primary macrophages occurs via two distinct intracellular nucleotide-sensing pathways responsible for induction of IFNs and inflammatory cytokine expression, respectively.Virus recognition is essential for activation of innate antiviral immune defense and the subsequent induction of acquired immunity. Conserved pathogen motifs, termed pathogen-associated molecular patterns (PAMPs), are recognized by pattern recognition receptors (PRRs). Virus-recognizing PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and a number of intracellular DNA receptors. Several TLRs have been attributed roles in the recognition of virus. TLR2 and TLR4 recognize viral surface structures (3, 6, 18, 31), TLR3 recognizes double-stranded RNA (dsRNA) (2), and TLR7/8 and TLR9 function as signaling receptors for viral single-stranded RNA (ssRNA) (8, 11, 21) and CpG DNA (12, 20), respectively.Within the cell, cytoplasmic RLRs RIG-I and MDA5 both recognize accumulation of virus-derived dsRNA; in addition, RIG-I recognizes 5′-triphosphated RNA (14, 27, 39, 40). In addition to the RLRs, a number of receptors recognize foreign DNA. Presently, three DNA receptors have been identified: Z-DNA binding protein 1 (ZBP-1, or DAI) (36) and RNA polymerase III (Pol III) (1, 4) both mediate interferon (IFN) and cytokine production, whereas the AIM2 inflammasome is involved in caspase 1 activation in response to cytoplasmic dsDNA (13).Herpes simplex virus type 1 (HSV-1) and HSV-2 are two closely related human DNA viruses associated with a number of serious diseases, including orofacial infections, encephalitis, and genital infections (34). Macrophages play an important role in the first line of defense against viral infection via production of IFNs, cytokines, and chemokines that regulate the progress of the virus infection and activate and support appropriate defense mechanisms (9, 10, 24).TLR2, TLR3, and TLR9 have been identified as mediators of proinflammatory cytokine production during HSV infections. TLR2 mediates an overzealous inflammatory cytokine response following HSV-1 infection in mice, promoting mononuclear cell infiltration of the brain and development of encephalitis (18). TLR3 mediates type I and III IFN production in human fibroblasts (41). TLR9 recognizes genomic DNA from HSV-1 and HSV-2 in murine plasmacytoid dendritic cells (DCs) (17, 20) and mediates tumor necrosis factor alpha (TNF-α) and CCL5 production in murine macrophages (22). Both TLR2 and TLR9 mediate recognition of HSV and cytokine production in murine conventional DCs (35). HSV is recognized by an RLR/MAVS-dependent mechanism in murine macrophages and mouse embryonic fibroblasts (MEFs) (5, 29, 30). Recent data suggest that RNA Pol III mediates IFN production following HSV-1 infection and transfection with HSV-1 DNA in macrophage-like RAW 264.7 cells (4). Finally, murine L929 fibroblast-like cells are moderately inhibited in their ability to produce IFN after HSV-1 infection when ZBP-1 is knocked down (19, 36). Thus, several PRRs have been reported to recognize HSV-1 in murine cells and different cell lines, but the pathways responsible for sensing this virus in human primary macrophages and their impact on cytokine expression have not previously been described.In this work, we investigate the recognition pathways underlying HSV-induced cytokine and chemokine expression in human primary macrophages. We demonstrate that HSV-1-induced IFN and cytokine expression is independent of TLR2 and TLR9 but highly dependent on virus replication and intracellular nucleotide recognition systems. Specifically, induction of IFNs is dependent on MAVS and MDA5, whereas TNF-α is induced by a novel intracellular nucleotide-sensing system.     

Acute Oxidative Stress Can Reverse Insulin Resistance by Inactivation of Cytoplasmic JNK

Chronic oxidative stress results in decreased responsiveness to insulin, eventually leading to diabetes and cardiovascular disease. Activation of the JNK signaling pathway can mediate many of the effects of stress on insulin resistance through inhibitory phosphorylation of insulin receptor substrate 1. By contrast, exercise, which acutely increases oxidative stress in the muscle, improves insulin sensitivity and glucose tolerance in patients with Type 2 diabetes. To elucidate the mechanism underlying the contrasting effects of acute versus chronic oxidative stress on insulin sensitivity, we used a cellular model of insulin-resistant muscle to induce either chronic or acute oxidative stress and investigate their effects on insulin and JNK signaling. Chronic oxidative stress resulted in increased levels of phosphorylated (activated) JNK in the cytoplasm, whereas acute oxidative stress led to redistribution of JNK-specific phosphatase MKP7 from the nucleus into the cytoplasm, reduction in cytoplasmic phospho-JNK, and a concurrent accumulation of phospho-JNK in the nucleus. Acute oxidative stress restored normal insulin sensitivity and glucose uptake in insulin-resistant muscle cells, and this effect was dependent on MKP7. We propose that the contrasting effects of acute and chronic stress on insulin sensitivity are driven by changes in subcellular distribution of MKP7 and activated JNK.     

Variation in seed protein and isoenzyme patterns in Cucurbita cultivars

The genetic variability in the seed proteins and the enzyme alcohol dehydrogenase (ADH) in representative species of the genus Cucurbita was studied. The banding patterns were obtained by means of vertical block electrophoresis in polyacrylamide gel. A specific protein components and ADH isoenzymes were established in the polymorphic banding patterns which can be applied individually or in combination as potential biochemical markers for breeding purposes.     

Redox-regulated ion channel activity of a cysteine-containing gramicidin A analogue

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes: classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution, if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H2O2 led to an increase in the transmembrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.