Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action

Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1.     

Molecular analysis of PKU haplotypes in the population of southern Poland

Summary Out of a population of 138598 infants born in southern Poland between 1987 and 1989, and screened for phenylketonuria (PKU), 28 cases were ascertained giving an incidence of 1 in 5000. DNA from 22 of these probands and their parents was isolated and eight polymorphic restriction sites were analyzed within the phenylalanine hydroxylase gene region. Twenty-one different haplotypes (HT) were revealed, five of them representing new categories. The most common haplotypes among those carrying normal alleles were: HT1 (27.3%) and HT4 (11.4%). Within the group of haplotypes with mutant alleles the most frequent was HT2 (56.8%), whereas the frequency of this haplotype in other European populations, such as French, Danish and German, ranged from 12% to 24%. HT3, being the most common in Danish (38%), and relatively frequent in the other western European (13–14%) populations, appeared to be very rare in our sample (2.3%). The mutation of codon 408 (exon 12, CT, ArgTrp), which has been described to be tightly linked to HT2, was tested on amplified DNA by dot-blot hybridization. This mutation was found in 25 out of 44 proband chromosomes. In one case it was linked to HT5, in the remaining 24 to HT2. Our results confirm molecular heterogeneity of PKU haplotypes, as well as their significant interpopulation variation.     

Structural characterization of binding of Cu(II) to tau protein

Transition metals have been frequently recognized as risk factors in neurodegenerative disorders, and brain lesions associated with Alzheimer's disease are rich in Fe(III), Zn(II), and Cu(II). By using different biophysical techniques (nuclear magnetic resonance, circular dichroism, light scattering, and microcalorimetry), we have structurally characterized the binding of Cu(II) to a 198 amino acid fragment of the protein Tau that can mimic both the aggregation behavior and microtubule binding properties of the full-length protein. We demonstrate that Tau can specifically bind one Cu(II) ion per monomer with a dissociation constant in the micromolar range, an affinity comparable to the binding of Cu(II) to other proteins involved in neurodegenerative diseases. NMR spectroscopy showed that two short stretches of residues, (287)VQSKCGS (293) and (310)YKPVDLSKVTSKCGS (324), are primarily involved in copper binding, in agreement with mutational analysis. According to circular dichroism and NMR spectroscopy, Tau remains largely disordered upon binding to Cu(II), although a limited amount of aggregation is induced.     

Partial Purification and Characterization of Indol-3-Ylacetylglucosemyo-Inositol Indol-3-Ylacetyltransferase (Indoleacetic Acid-Inositol Synthase)

A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-β-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-β-d-glucose is, K_m = 30 micromolar, and for myo-inositol is, K_m = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.     

Calcium dysregulation in Alzheimer's disease

Alzheimer disease (AD) is the most common form of adult dementia. Its pathological hallmarks are synaptic degeneration, deposition of amyloid plaques and neurofibrillary tangles, leading to neuronal loss. A few hypotheses have been proposed to explain AD pathogenesis. The beta-amyloid (Abeta) and hyperphosphorylated tau hypotheses suggest that these proteins are the main players in AD development. Another hypothesis proposes that the dysregulation of calcium homeostasis may be a key factor in accelerating other pathological changes. Although Abeta and tau have been extensively studied, recently published data provide a growing body of evidence supporting the critical role of calcium signalling in AD. For example, presenilins, which are mutated in familial cases of AD, were demonstrated to form low conductance calcium channels in the ER and elevated cytosolic calcium concentration increases amyloid generation. Moreover, memantine, an antagonist of the NMDA-calcium channel receptor, has been found to have a beneficial effect for AD patients offering novel possibilities for a calcium signalling targeted therapy of AD. This review underscores the growing importance of calcium ions in AD development and focuses on the relevant aspects of calcium homeostasis.     

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.     

Fibroblast-like synoviocytes from rheumatoid arthritis patients express functional IL-15 receptor complex: endogenous IL-15 in autocrine fashion enhances cell proliferation and expression of Bcl-x(L) and Bcl-2

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-15Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-15Ralpha chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.     

Prognostic Significance of ESR1 Amplification and ESR1 PvuII,CYP2C19*2, UGT2B15*2 Polymorphisms in Breast Cancer Patients

Introduction

Amplification of the ESR1 gene, coding for estrogen receptor alpha, was shown to predict responsiveness to tamoxifen, however its prognostic impact in breast cancer patients has not been thoroughly investigated. Other factors that could contribute to responsiveness to tamoxifen treatment are polymorphisms in ESR1 gene and genes involved in tamoxifen metabolism.The aim of this study was to assess the prognostic role of ESR1 gene dosage in a consecutive group of breast cancer patients and to correlate this feature with clinico-pathological factors. Additionally, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphisms were analyzed in the tamoxifen-treated subgroup of patients.

Materials and Methods

Primary tumor samples from 281 stage I-III consecutive breast cancer patients were analyzed for ESR1 gene dosage using real-time PCR with locked nucleic acids hydrolysis probes. In the tamoxifen-treated subgroup of patients, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphism in leukocytes genomic DNA were analyzed. Results were correlated with clinico-pathological factors and with disease-free survival (DFS) and overall survival (OS).

Results

ESR1 amplification (with a cut-off level of 2.0) was found in 12% of the entire group of breast cancer patients, and in 18% of the ER-negative subgroup. This feature was associated with decreased DFS both in the entire group (P=0.007) and in the ER-negative subgroup (P=0.03), but not in the tamoxifen-treated patients.Patients with ESR1 PvuII wt/wt genotype and at least one UGT2B15 wt allele had a worse DFS (P=0.03) and showed a trend towards decreased Os (P=0.08) in comparison to patients with ESR1 PvuII wt/vt or vt/vt genotype and UGT2B15 *2/*2 genotype.

Conclusions

ESR1 amplification can occur in ER-negative tumors and may carry poor prognosis. In the tamoxifen-treated subgroup, poor prognosis was related to the combined presence of ESR1 PvuII wt/wt and UGT2B15wt/wt or wt/*2 genotype.