Erythropoietin Receptor Expression Is a Potential Prognostic Factor in Human Lung Adenocarcinoma

Recombinant human erythropoietins (rHuEPOs) are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR) signaling in human non-small cell lung cancer (NSCLC) also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III–IV adenocarcinoma (ADC) and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC) proliferation was determined by 5-bromo-2’-deoxy-uridine (BrdU) incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine) did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.     

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or,a novel flavoheme reductase

NADH cytochrome b₅ oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.     

Genetic characterization and population bottleneck in the Hucul horse based on microsatellite and mitochondrial data

The aim of this work was to gather information about the origin and genetic characterization of the Central European Hucul horse based on 71 horses using 17 microsatellites and the D‐loop region of mtDNA. Their genetic relationship to the Polish Konik (N = 7), German (N = 4) and Hungarian wild Przewalski horses (N = 4) and 200 horse sequences from GenBank was also analysed. Both microsatellite and mtDNA analysis showed a high genetic variation in the Hucul. A total of 130 alleles were detected, the mean number of observed alleles per microsatellite was 7.647, and the number of effective alleles was 4.401. The average observed and expected heterozygosity were 0.706 and 0.747, respectively. The high heterozygosity values and Wright's fixation index (F_IS) (?0.128) indicated a low level of inbreeding, low or no selection pressure, and large number of alleles. mtDNA analysis revealed 18 haplotypes for the Hucul population with a total of 23 variable sites. Haplotype and nucleotide diversities were 0.935 ± 0.011 and 0.022 ± 0.012, respectively. Neutrality tests (Tajima's D and Fu's Fs) were non‐significant, and mismatch distribution was ragged, indicating that the Hucul population is in genetic equilibrium. The most frequent mtDNA D‐loop region belonged to haplogroup A (48%), which was also present in Przewalski Wild horse samples, while Polish Konik samples belonged to three haplotypes and C, F, and G haplogroups. Large and significant pairwise Φ_ST values along with a small number of common haplotypes indicated a low level of gene flow and lack of genetic structure among the three studied breeds (Hucul, Konik, and Przewalski Wild horse). The present work contributes to our knowledge of the genetic diversity of the Hucul horse and helps to define its genetic conservation. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 54–65.     

Maternal genomic variability of the wild boar (Sus scrofa) reveals the uniqueness of East‐Caucasian and Central Italian populations

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian (n = 8), European (n = 64), and North African (n = 5) wild boar. Our analyses provided evidence of unique lineages in the East‐Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin.     

Application of capillary zone electrophoresis to the analysis and to a stability study of cephalosporins

1-Methylpropyl-2-imidazolyl disulfide (MID) is a novel antitumor agent currently in Phase I clinical trials. The chromatographic behavior of MID and its potential impurity, degradation product, and metabolite 2-mercaptoimidazole (2MI) was studied under reversed-phase (RP) and normal-phase (NP) conditions. Both RP- and NP-HPLC separation methods were developed. RP-HPLC was validated as a stability-indicating assay for MID. NP-HPLC retained both MID and 2MI and pending further validation, could prove useful in the study of MID pharmacokinetics.     

Interleukin-6 N-terminal peptides modulate the expression of junB protooncogene and the production of fibrinogen in HepG2 cells

Interleukin-6 (IL-6) is a helical cytokine exerting pleiotropic activities including the regulation of hematopoiesis, B cell activation and acute-phase reaction. The structure-function relationship of the molecule is the subject of intensive investigation using point and deletion mutants. Our objective was to analyse the role of the N-terminal 18-46 region in IL-6-mediated expression of junB protooncogene and fibrinogen production, reflecting the acute phase response, with synthetic overlapping peptides. mRNA expression of junB was monitored by competitive RT-PCR, while sandwich ELISA was used for the detection of fibrinogen in the supernatant of HepG2 human hepatoma cells. We found that even short synthetic octapeptides can be stimulatory (in the absence of IL-6) or inhibitory (in the presence of IL-6) in both assays. To establish the molecular mechanism by which synthetic peptides exert their biological effects electromobility shift assay was carried out using HepG2 nuclear extracts. Peptides inducing junB expression initiate gel shifts of STAT3/DNA complexes, which may indicate the involvement of this signal transduction pathway. Circular dicroism spectroscopy data suggest that 8-11-mer peptides representing different parts of the 18-46 region have a marked tendency to adopt ordered conformations in a water/trifluoroethanol (1:1 v/v) mixture. Competition studies with rhIL-6 and selected fluorophore-labelled peptides indicate the presence of more than one binding site on soluble IL-6 receptor. Considering the possible multiple etiologic role of IL-6 in the pathogenesis of various diseases, these peptides could be useful for dissection of IL-6 related biological effects.     

Cloning and heterologous expression of a beta-D-mannosidase (EC 3.2.1.25)-encoding gene from Thermobifida fusca TM51

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.