Bioenergetics of the Calf Muscle in Friedreich Ataxia Patients Measured by 31P-MRS Before and After Treatment with Recombinant Human Erythropoietin

Friedreich ataxia (FRDA) is caused by a GAA repeat expansion in the FXN gene leading to reduced expression of the mitochondrial protein frataxin. Recombinant human erythropoietin (rhuEPO) is suggested to increase frataxin levels, alter mitochondrial function and improve clinical scores in FRDA patients. Aim of the present pilot study was to investigate mitochondrial metabolism of skeletal muscle tissue in FRDA patients and examine effects of rhuEPO administration by phosphorus 31 magnetic resonance spectroscopy (31P MRS). Seven genetically confirmed FRDA patients underwent 31P MRS of the calf muscles using a rest-exercise-recovery protocol before and after receiving 3000 IU of rhuEPO for eight weeks. FRDA patients showed more rapid phosphocreatine (PCr) depletion and increased accumulation of inorganic phosphate (Pi) during incremental exercise as compared to controls. After maximal exhaustive exercise prolonged regeneration of PCR and slowed decline in Pi can be seen in FRDA. PCr regeneration as hallmark of mitochondrial ATP production revealed correlation to activity of complex II/III of the respiratory chain and to demographic values. PCr and Pi kinetics were not influenced by rhuEPO administration. Our results confirm mitochondrial dysfunction and exercise intolerance due to impaired oxidative phosphorylation in skeletal muscle tissue of FRDA patients. MRS did not show improved mitochondrial bioenergetics after eight weeks of rhuEPO exposition in skeletal muscle tissue of FRDA patients.

Trial Registration

EU Clinical Trials Register 2008-000040-13     

Functionalized congeners of tyrosine-based P2X(7) receptor antagonists: probing multiple sites for linking and dimerization

Chemically funtionalized analogues of antagonists of the P2X(7) receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-L-tyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X(7) receptor function, consisting of inhibition of ATP-induced K(+) efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R(1)-Tyr(OR(2))-piperazinyl-R(3), in which three positions (R(1)-R(3)) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3- or 4-nitrophenyl group. The nitro groups were reduced using NaBH(4)-copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N(alpha)-3-aminobenzyloxycarbonyl functionalization was devised. The various positions of functionalization were compared for effects on biological potency, and the R(2) and R(3) positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R(2)-position were potent antagonists. In concentration-response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC(50) values of roughly 100 nM as antagonists of P2X(7) receptor-mediated K(+) flux.     

A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor of human leukemic lymphocytes

The P2X(7)R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X(7)R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X(7)R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X(7)R function was monitored by measuring ATP-induced Ca(2+) influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X(7) allelic variant. Ca(2+) influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca(2+) flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X(7)R confirmed an increased ATP-dependent activation of the P2X(7) 489T mutant with respect to the wild type receptor, as assessed by both by [Ca(2+)](i) influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X(7)R.     

Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer

The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.     

A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum: MOLECULAR MODELING AND FUNCTIONAL PROPERTIES*

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O₂ with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.     

On Mellitella stokesii and Amphipholis geminata (Echinodermata), from an intertidal flat in the upper Gulf of Nicoya estuary, Pacific, Costa Rica

Information on changes over time in the biodiversity and ecology of tropical systems is of increasing importance against the background of local, regional, and global dynamics. This study aimed to contribute with long-term data on the abundance fluctuations of two species of echinoderms from a tropical tidal flat. From February 1984 to December 1986, March 1985 to April 1987, and July 1994 to September 1996 (total: 76 dates), core samples (17.7 cm2, 15 cm deep) were collected at low tide at a mud-sand flat in the mid upper Gulf of Nicoya estuary, Costa Rica, as part of a survey of the benthic community. Among more than 100 taxa, the burrowing brittle star, Amphipholis geminata, and the sand dollar, Mellitella stokesii, consistently appeared in the samples over the study period. A total of 63 M. stokesii and 149 A. geminata were collected. The 43 sand dollars found during 1984-1985, give an approximate density of 35 ind./m2, which is within the range reported for this species. M. stokesii was almost absent from 1994 to 1996, while A. geminata had its peak of abundance at the end of 1995. Low abundances of the sand dollar during the rainy seasons (May-November) and slight increments in dry seasons (December-April) cores, also agrees with a report from the region. A. geminata also presented an irregular pattern of abundance, with slight increases at the end of the rainy seasons or during the dry seasons, when higher salinities are more suitable for echinoderms. The patchy spatial distribution of both species makes difficult the detection of patterns with a corer. Nevertheless, this information is unique due to its extensive time coverage and provides a baseline for future surveys designed specifically for the study of tropical intertidal estuarine echinoderms.     

Functional and physical interaction of blue- and red-light sensors in Aspergillus nidulans

Light sensing is very important for organisms in all biological kingdoms to adapt to changing environmental conditions. It was discovered recently that plant-like phytochrome is involved in light sensing in the filamentous fungus Aspergillus nidulans[1]. Here, we show that phytochrome (FphA) is part of a protein complex containing LreA (WC-1) and LreB (WC-2) [2, 3], two central components of the Neurospora crassa blue-light-sensing system. We found that FphA represses sexual development and mycotoxin formation, whereas LreA and LreB stimulate both. Surprisingly, FphA interacted with LreB and with VeA, another regulator involved in light sensing and mycotoxin biosynthesis. LreB also interacted with LreA. All protein interactions occurred in the nucleus, despite cytoplasmic subfractions of the proteins. Whereas the FphA-VeA interaction was dependent on the presence of the linear tetrapyrrole in FphA, the interaction between FphA and LreB was chromophore independent. These results suggest that morphological and physiological differentiations in A. nidulans are mediated through a network consisting of FphA, LreA, LreB, and VeA acting in a large protein complex in the nucleus, sensing red and blue light.