12 research outputs found
Biophysical and electrochemical studies of protein-nucleic acid interactions
This review is devoted to biophysical and electrochemical methods used for studying protein-nucleic acid (NA) interactions. The importance of NA structure and protein-NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein-DNA and protein-RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein-NA interactions. Recent applications of electrochemical methods in studies of protein-NA interactions are discussed in detail
The Transcription Factor AmrZ Utilizes Multiple DNA Binding Modes to Recognize Activator and Repressor Sequences of Pseudomonas aeruginosa Virulence Genes
AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation
Patients with chronic idiopathic neutropenia of adults have increased serum concentrations of inflammatory cytokines and chemokines
Serum levels of inflammatory cytokines and chemokines were measured in
132 patients with chronic idiopathic neutropenia of adults (CINA) and 34
healthy Volunteers (controls) using commercially available micro-ELISA
determination kits, We found that serum interleukin-1 beta (IL-1 beta),
tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6),
transforming growth factor-beta (1) (TGF-beta (1)), and soluble tumor
necrosis factor receptor p55 (sTNF-RI) were all significantly increased
in CINA patients compared to controls. Individual cytokine values
inversely correlated with the number of circulating neutrophils. Serum
levels of interleukin-8 (IL-8) and RANTES, two potent chemokines for
neutrophils and lymphocytes, respectively, were also significantly
increased in the group of patients and they inversely correlated with
the number of circulating neutrophils, Contrarily, serum levels of
interleukin-l (IL-4), interferon-gamma (IFN gamma), soluble CD23
(sCD23), and soluble interleukin-a receptor (sIL-2R) did not show any
significant change in the patients studied, We assume that CINA patients
have increased serum concentrations of inflammatory cytokines and
chemokines mainly produced by activated macrophages, while they disclose
normal levels of inflammatory molecules mainly released from activated
lymphocytes. These findings provide further evidence for an underlying
low-grade chronic inflammatory process in CINA patients, as we
previously have suggested, If this chronic inflammation is really the
cause of the disorder or it simply represents the result of neutropenia
remains to be elucidated. (C) 2000 Wiley-Liss, Inc
The oestrogen receptor codon 10 polymorphism detected in breast cancer is also present in non-malignant cells
The effect of oestrogens on oestrogen-receptive organs and cells is
mediated via intracellular receptors (ER alpha and ER beta). Oestrogen
receptor gene polymorphisms in the region encoding the N-terminal
portion of the protein are reportedly associated with pathological
conditions including breast cancer, hypertension, spontaneous abortion
and coronary heart disease. A silent mutation in codon 10 of exon 1,
detected in ER-negative and ER-positive human breast cancer cell lines,
in breast tumors and blood DNA from breast cancer patients, has been
recognized as a polymorphic site. In this study we examined. by
denaturing gradient-gel electrophoresis and DNA sequence analysis, the
possible presence of a codon 10 polymorphic site in normal oestrogen
target organs and cells such as the uterus (myometrium and endometrium),
in the placenta and peripheral blood mononuclear cells and in a benign
uterus tumour (leiomyoma). We have detected ER codon 10 polymorphism in
these samples and, have compared them to those observed in breast cancer
samples. All tissues and cells studied were homozygous for the wild-type
gene, and were heterozygous as well as homozygous for the
codon-10-variant type. These results indicate that the presence of the
codon-10-variant type is not a characteristic of breast cancer. Out
current findings suggest that further investigations are warranted to
elucidate the possible linkage of ER codon 10 polymorphism to
physiological and pathological conditions
Detection of oestrogen receptor variants in endometrium, myometrium, leiomyoma and peripheral blood mononuclear cells: comparison to variants present in breast cancer
Oestradiol has mitogenic and regulatory effects on various organs and
cells, mediated mainly by its nuclear receptor (ER). The presence of
aberrant ER forms in Oestrogen-dependent tumours has been discussed in
correlation with tumour progression. ER variants, generated by
alternative splicing, have been detected in human breast cancer, but
also in normal mammary glands, therefore their role in tumorigenesis has
been questioned. We have investigated, by the use of the reverse
transcription polymerase chain reaction amplification technique, the
possible existence of ER variants in other normal oestrogen target
organs and cells, such as uterus (myometrium and endometrium), in
peripheral blood mononuclear cells and in a benign uterus tumour
(leiomyoma). We have detected variant ER in these samples and have
compared the variant profile to that observed in breast cancer. All
tissues and cells studied expressed both wild-type ER and variant
species. Variant forms encompassed ER with deletions of exons 2, 5 and
7. Variants with exon 5 deleted were detected only in peripheral blood
mononuclear cells and in breast cancer. Variants with exons 2 and 7
deleted were present in all specimens tested. These results corroborate
previous findings that the presence of ER variants is not a
characteristic of breast cancer. The physiological significance and
possible clinical relevance of the variant ER forms remain to be
elucidated
Detection of oestrogen receptor variants in endometrium, myometrium, leiomyoma and peripheral blood mononuclear cells: comparison to variants present in breast cancer
Journal URL: http://www.springerlink.com/content/101168
Levels of serum cytokines and acute phase proteins in patients with essential and cancer-related thrombocytosis
Essential thrombocytosis (ET) is a myeloproliferative disorder resulting in an increased production of abnormal platelets. Reactive thrombocytosis (RT) is occasionally observed in clinical situations including chronic inflammation and malignancy. The aim of the present study was to evaluate the discriminatory efficiency of various laboratory tests in patients with ET and cancer-related RT. Forty-five patients with ET, 52 patients with RT, and 25 age-matched normal individuals comprised the study population. Plasma interleukin-1 alpha (IL-1a), IL-2, IL-6, tumor necrosis factor alpha (TNF-a), platelets, hematocrit, hemoglobin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), lactate dehydrogenase (LDH) and ferritin were determined. We found increased levels of territin, LDH, CRP, ESR, IL-1a, and IL-6 in RT compared with ET (p < 0.01 to p < 0.0005). Hemoglobin, hematocrit, and platelets were significantly lower in RT than in ET (p < 0.0005). Furthermore, ferritin and ESR were negatively correlated with Hct, hemoglobin, and TNF-a, whereas ferritin was positively correlated with ESR, IL-1a, IL-6, and CRP, and IL-1a was positively correlated with IL-6. We consider that the aforementioned parameters should be included in the investigation of unexplained thrombocytosis for the differentiation of essential from cancer related thrombocytosis
Acetyl Cholinesterase Inhibitors and Cell-Derived Peripheral Inflammatory Cytokines in Early Stages of Alzheimer's Disease
Background Clinical and preclinical studies firmly support the involvement of the inflammation in the pathogenesis of Alzheimer's disease (AD). Despite acetylcholinesterase inhibitors (AChEI) being widely used in AD patients, there is no conclusive evidence about their impact on the inflammatory response. Methods This study investigates peripheral proinflammatory cytokines (interferon gamma [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukins 1β [IL-1β] and 6 [IL-6]) by firstly comparing peripheral blood mononuclear cell (PBMC)-derived secretion in drug-naïve and AChEI-treated AD patients versus healthy controls. A subset of those drug-naïve AD patients, who were prescribed the AChEI donepezil, was followed-up for 6 months to investigate if donepezil suppresses proinflammatory cell-derived cytokine secretion. Results Patients with AD showed higher levels of PBMC-derived proinflammatory cytokines (IFN-γ, TNF-α, IL-1β, and IL-6) in comparison with healthy controls. On reexamination, previously drug-naïve AD patients who received donepezil treatment for 6 months displayed a decrease in cell-derived IFN-γ, TNF-α, IL-1β, and IL-6. Conclusions Proinflammatory PBMC-derived cytokines were increased in patients with AD in comparison with healthy controls and donepezil-reduced proinflammatory cytokines when examining drug-naïve AD patients before and after AChEI treatment. Copyright © Wolters Kluwer Health, Inc. All rights reserved