Amperometric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) film

Organic peroxides, t-butyl hydroperoxide, 2-butanone peroxide, cumene hydroperoxide and t-butyl peracetate, were determined by an amperometric enzyme electrode. The enzyme electrode was prepared through electrostatic immobilization of horseradish peroxidase (HRP) in a polyvinylferrocenium (PVF) film. A PVF(+)ClO(4)(-) film was coated on a Pt foil at +0.70 V by electrooxidation of polyvinylferrocene in methylene chloride with 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode PVF(+)HRP(-) was prepared by anion-exchange in a solution of HRP(-) in 0.05 M phosphate buffer at pH 8.5. FTIR spectroscopy was used to identify PVF, PVF(+)ClO(4)(-), and PVF(+)HRP(-). The immobilized amount of the enzyme in the film was determined by UV spectroscopy. The effects of the polymeric film thickness, bulk enzyme concentration used in the immobilization treatment and the temperature on the performance of enzyme electrode were investigated. The inhibitory effect of oxygen was also examined. Linearities, lower detection limits, active life times and sensitivities of the electrode were determined for each peroxide.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity

Background

Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, “normal-like”, and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity.

Methodology/Principal Findings

A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21^Cip1 correlated with senescence in these cell lines. p21^Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and “normal-like” tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice.

Conclusions/Significance

Luminal A and “normal-like” breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.     

Mutation in exon 1f of PLEC, leading to disruption of plectin isoform 1f, causes autosomal-recessive limb-girdle muscular dystrophy

Limb-girdle muscular dystrophy (LGMD) is a genetically heterogeneous group of inherited muscular disorders manifesting symmetric, proximal, and slowly progressive muscle weakness. Using Affymetrix 250K SNP Array genotyping and homozygosity mapping, we mapped an autosomal-recessive LGMD phenotype to the telomeric portion of chromosome 8q in a consanguineous Turkish family with three affected individuals. DNA sequence analysis of PLEC identified a homozygous c.1_9del mutation containing an initiation codon in exon 1f, which is an isoform-specific sequence of plectin isoform 1f. The same homozygous mutation was also detected in two additional families during the analysis of 72 independent LGMD2-affected families. Moreover, we showed that the expression of PLEC was reduced in the patient's muscle and that there was almost no expression for plectin 1f mRNA as a result of the mutation. In addition to dystrophic changes in muscle, ultrastructural alterations, such as membrane duplications, an enlarged space between the membrane and sarcomere, and misalignment of Z-disks, were observed by transmission electron microscopy. Unlike the control skeletal muscle, no sarcolemmal staining of plectin was detected in the patient's muscle. We conclude that as a result of plectin 1f deficiency, the linkage between the sarcolemma and sarcomere is broken, which could affect the structural organization of the myofiber. Our data show that one of the isoforms of plectin plays a key role in skeletal muscle function and that disruption of the plectin 1f can cause the LGMD2 phenotype without any dermatologic component as was previously reported with mutations in constant exons of PLEC.     

Three-dimensional biofilm image reconstruction for assessing structural parameters

Parameters representing three-dimensional (3D) biofilm structure are quantified from confocal laser-scanning microscope (CLSM) images. These 3D parameters describe the distribution of biomass pixels within the space occupied by a biofilm; however, they lack a direct connection to biofilm activity. As a result, researchers choose a handful of parameters without there being a consensus on a standard set of parameters. We hypothesized that a select 3D parameter set could be used to reconstruct a biofilm image and that the reconstructed and original biofilm images would have similar activities. To test this hypothesis, an algorithm was developed to reconstruct a biofilm image with parameters identical to those of the original CLSM image. We introduced an objective method to assess the reconstruction algorithm by comparing the activities of the original and reconstructed biofilm images. We found that biofilm images with identical structural parameters showed nearly identical activities and substrate concentration profiles. This implies that the set containing all common structural parameters can successfully describe biofilm structure. This finding is significant, as it opens the door to the next step, of finding a smaller standard set of biofilm structural parameters that can be used to compare biofilm structure.     

Diet-induced obesity suppresses ghrelin in rat gastrointestinal tract and serum

The aims of the present study were to examine ghrelin expression in serum and gastrointestinal tract (GIT) tissues, and to measure tissue ghrelin levels and obesity-related alterations in some serum biochemical variables in rats with diet-induced obesity (DIO). The study included 12 male rats, 60 days old. The rats were randomly allocated to two groups (n = 6). Rats in the DIO group were fed a cafeteria-style diet to induce obesity, while those in the control group were fed on standard rat pellets. After a 12 week diet program including an adaptation period all rats were decapitated, tissues were individually fixed, ghrelin expression was examined by immunohistochemistry , and tissue and serum ghrelin levels were measured by radioimmunoassay. Serum biochemical variables were measured using an autoanalyzer. When the baseline and week 12 body mass index and GIT ghrelin expression were compared between DIO and control rats, BMI had increased and ghrelin expression decreased due to obesity. The RIA results were consistent with these findings. Serum glucose, LDL cholesterol, and total cholesterol levels were elevated and HDL cholesterol significantly decreased in the DIO group. A comparison of GIT tissues between the control and obese groups demonstrated that ghrelin was decreased in all tissues of the latter. This decrease was brought about a decline in the circulating ghrelin pool. This suggests that rather than being associated with a change in a single tissue, obesity is a pathological condition in which ghrelin expression is changed in all tissues.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

G-308A TNF-α polymorphism is associated with an increased risk of hepatocellular carcinoma in the Turkish population: Case-control study

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that may act as an endogenous tumor promoter. A genetic polymorphism of TNF-α gene at position ?308 promoter region is involved in the regulation of expression level and has been found to be associated with susceptibility to various types of cancer. Methods: To determine the association of the TNF-α gene G-308A polymorphism on the risk of hepatocellular carcinoma (HCC) in a Turkish population, a hospital-based case-control study was designed consisting of 110 diagnosis subjects with hepatocellular carcinoma and 110 cancer-free control subjects matched on age, gender, smoking and alcohol status. The genotype frequency of this polymorphism was determined by using a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. Results: The distribution G-308A genotype was significantly associated with the risk of HCC (p < 0.001, odds ratio [OR] = 4.75, 95% confidence interval [CI] = 2.25–9.82 for ?308 AA/GA genotypes versus GG genotype). Conclusion: We suggested that the presence of the high producer allele ?308A in the TNF-α gene appears to be associated with an increased risk for the development of HCC in Turkish population.     

Photophysical study of photoinduced electron transfer in a bis-thiophene substituted peryleneimide.

Based on femtosecond time-resolved spectroscopy and single photon timing experiments, intramolecular photoinduced charge transfer has been investigated in two systems containing a peryleneimide chromophore (P) and thiophene (T) groups. The first compound bearing a single thiophene ring (PT1) is used as model and shows a behavior similar to P, studied previously, while in the compound with two thiophene rings attached (PT2) electron transfer from the thiophene donor to the peryleneimide acceptor is observed in benzonitrile. Femtosecond fluorescence upconversion and femtosecond transient absorption experiments in benzonitrile indicate that this ion-pair state formation occurs in 19 ps. This ion-pair state then decays with two time constants of 1400 and 820 ps, probably corresponding to different conformations of the thiophene rings.     

Network analyses identify liver‐specific targets for treating liver diseases

We performed integrative network analyses to identify targets that can be used for effectively treating liver diseases with minimal side effects. We first generated co‐expression networks (CNs) for 46 human tissues and liver cancer to explore the functional relationships between genes and examined the overlap between functional and physical interactions. Since increased de novo lipogenesis is a characteristic of nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC), we investigated the liver‐specific genes co‐expressed with fatty acid synthase (FASN). CN analyses predicted that inhibition of these liver‐specific genes decreases FASN expression. Experiments in human cancer cell lines, mouse liver samples, and primary human hepatocytes validated our predictions by demonstrating functional relationships between these liver genes, and showing that their inhibition decreases cell growth and liver fat content. In conclusion, we identified liver‐specific genes linked to NAFLD pathogenesis, such as pyruvate kinase liver and red blood cell (PKLR), or to HCC pathogenesis, such as PKLR, patatin‐like phospholipase domain containing 3 (PNPLA3), and proprotein convertase subtilisin/kexin type 9 (PCSK9), all of which are potential targets for drug development.