Identification and characterization of two related murine genes, Eat2a and Eat2b, encoding single SH2-domain adapters

Human EAT-2 (SH2D1B) and SLAM-associated protein (SAP) (SH2D1A) are single SH2-domain adapters, which bind to specific tyrosine residues in the cytoplasmic tail of six signaling lymphocytic activation molecule (SLAM) (SLAMF1)-related receptors. Here we report that, unlike in humans, the mouse and rat Eat2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similarly, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cells, CD8⁺ T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-2B coimmunoprecipitated with mouse SLAM in transfected cells and augmented tyrosine phosphorylation of the cytoplasmic tail of SLAM. Both EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, Lck, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Taken together, the data suggest that both EAT-2A and EAT-2B are adapters that recruit Src kinases to SLAM family receptors using a mechanism that is distinct from that of SAP. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users. S. Calpe and E. Erdős contributed equally to this work     

Light-sensitive coupling of rhodopsin and melanopsin to G(i/o) and G(q) signal transduction in Caenorhabditis elegans

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.     

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.     

The Oxidative Stress May be Induced by the Elevated Homocysteine in Schizophrenic Patients

The mechanisms of oxidative stress in schizophrenic patients are not fully understood. In the present study, we investigated the effect of elevated level of homocysteine (Hcys) on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in plasma, the level of carbonyl groups in plasma proteins, as well as the amount of 3-nitrotyrosine in plasma proteins isolated from schizophrenic patients. Patients hospitalised in I and II Psychiatric Department of Medical University in Lodz, Poland were interviewed with special questionnaire (treatment, course of diseases, dyskinesis and other EPS). According to DSM-IV criteria all patients had diagnosis of paranoid type. They were treated with antipsychotic drugs (clozapine, risperidone, olanzapine). Mean time of schizophrenia duration was about 5 years. High-performance liquid chromatography was used to analyse the total level of homocysteine in plasma. Levels of carbonyl groups and 3-nitrotyrosine residues in plasma proteins were measured by ELISA and a competition ELISA, respectively. The lipid peroxidation in plasma was measured by the level of TBARS. Our results showed that in schizophrenic patients the amount of homocysteine in plasma was higher in comparison with the control group. We also observed a statistically increased level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine in plasma proteins from schizophrenic patients. Moreover, our experiments indicate that the correlation between the increased amount of homocysteine and the oxidative stress exists. Considering the data presented in this study, we suggest that the elevated Hcys in schizophrenic patients may stimulate the oxidative stress.     

Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples

Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.     

Analysis of mercury levels in historical bone material from syphilitic subjects--pilot studies (short report)

The aim of the present work is to study the remains of seven individuals with typical symptoms of tertiary syphilis in terms of mercury content in bones, thereby verifying whether they were subjected to treatment and, if they were, how long their organisms were exposed to toxic mercury fumes. Mercury was used, mainly in the Middle Ages and in the early modern period, as a preventive measure in case of individuals suffering from syphilis, a venereal disease, and also leprosy. Syphilitic patients treated this way should demonstrate increased concentration of mercury in their bones. The skeletons studied in the present work originate from various archaeological sites in southern and north-central Poland. The analyses concerned individuals with diagnosed syphilis as well as healthy individuals who constituted the control group. The analyses were performed by the LA-ICP-MS technique, with the use of laser Nd: YAG, Macro, 266 nm, New Wave, USA, coupled with Spectrometer Elan DRC-e Perkin Elmer, USA. The content analysis of the studied bone material revealed with high probability that the contact method of mercurial treatment was used only in the case of two women from north-central Poland, deceased at the turn of the 15th century at the earliest.     

Alcohol abuse and glycoconjugate metabolism

The relationship between alcohol consumption and glycoconjugate metabolism is complex and multidimensional. This review summarizes the advances in basic and clinical research on the molecular and cellular events involved in the metabolic effects of alcohol on glycoconjugates (glycoproteins, glycolipids, and proteoglycans). We summarize the action of ethanol, acetaldehyde, reactive oxygen species (ROS), nonoxidative metabolite of alcohol--fatty acid ethyl esters (FAEEs), and the ethanol-water competition mechanism, on glycoconjugate biosynthesis, modification, transport and secretion, as well as on elimination and catabolism processes. As the majority of changes in the cellular metabolism of glycoconjugates are generally ascribed to alterations in synthesis, transport, glycosylation and secretion, the degradation and elimination processes, of which the former occurs also in extracellular matrix, seem to be underappreciated. The pathomechanisms are additionally complicated by the fact that the effect of alcohol intoxication on the glycoconjugate metabolism depends not only on the duration of ethanol exposure, but also demonstrates dose- and regional-sensitivity. Further research is needed to bridge the gap in transdisciplinary research and enhance our understanding of alcohol- and glycoconjugate-related diseases.     

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP+ oxidoreductase

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP(+) oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9262-z) contains supplementary material, which is available to authorized users.     

Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.