ACTB,CDKN1B,GAPDH, GRB2, RHOA and SDCBP Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology

BackgroundNeuroendocrine lung cancer (NELC) represents 25% of all lung cancer cases and large patient collectives exist as formalin-fixed, paraffin-embedded (FFPE) tissue only. FFPE is controversially discussed as source for molecular biological analyses and reference genes for NELC are poorly establishes.ResultsRNA integrity (RIN) ranged from 1.8 to 2.6 and concentrations from 34 to 2,109 ng/μl. However, the nCounter technology gave evaluable results for all samples tested. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP were identified as constantly expressed genes with high stability (M-)values according to geNorm, NormFinder and coefficients of correlation.ConclusionFFPE-derived mRNA is suitable for molecular biological investigations via the nCounter technology, although it is highly degraded. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP are potent reference genes in neuroendocrine tumors of the lung.     

Evaluation of citrate synthase activity in brain of rats submitted to an animal model of mania induced by ouabain

Bipolar disorder (BD) is a psychiatric disorder characterized by alternating episodes of mania and depression. The intracerebroventricular (i.c.v) administration of ouabain (a Na⁺/K⁺-ATPase inhibitor) in rats has been used as an animal model of mania, because present face, construct and predictive validities. Several studies strongly suggest that mitochondrial dysfunction play a central role in the pathophysiology of BD. Citrate synthase (CS) is an enzyme localized in the mitochondrial matrix and represents one of the most important steps of Krebs cycle. The aim of this study was to investigate CS activity in brain of rats after the administration of ouabain. Adult male Wistar rats received a single i.c.v. administration of ouabain (10^?2 and 10^?3 M) or vehicle (control group). Locomotor activity was measured using the open field task. CS activity was measured in the brain of rats immediately (1 h) and 7 days after ouabain administration. Our results showed that spontaneous locomotion was increased 1 h after ouabain administration, and that the hyperlocomotion persists 7 days after the administration. Moreover, CS activity was inhibited immediately after the administration of ouabain in the prefrontal cortex at the doses of 10^?3 and 10^?2 M. This inhibition remains by 7 days after the administration of ouabain. On the other hand, it was not observed any difference in CS activity in the hippocampus and striatum. Considering that inhibition of CS activity may reflect a mitochondrial dysfunction, it is tempting to speculate that the reduction of brain energy metabolism might be related to the pathophysiology of BD.     

Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation

TDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3′-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3′-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels within cells, an essential regulatory process in maintaining cellular viability.     

Effects of acute and chronic administration of fenproporex on DNA damage parameters in young and adult rats

Obesity is a chronic and multifactorial disease, whose prevalence is increasing in many countries. Pharmaceutical strategies for the treatment of obesity include drugs that regulate food intake, thermogenesis, fat absorption, and fat metabolism. Fenproporex is the second most commonly consumed amphetamine-based anorectic worldwide; this drug is rapidly converted in vivo into amphetamine, which is associated with neurotoxicity. In this context, the present study evaluated DNA damage parameters in the peripheral blood of young and adult rats submitted to an acute administration and chronic administration of fenproporex. In the acute administration, both young and adult rats received a single injection of fenproporex (6.25, 12.5 or 25 mg/kg i.p.) or vehicle. In the chronic administration, both young and adult rats received one daily injection of fenproporex (6.25, 12.5, or 25 mg/kg i.p.) or Tween for 14 days. 2 h after the last injection, the rats were killed by decapitation and their peripheral blood removed for evaluation of DNA damage parameters by alkaline comet assay. Our study showed that acute administration of fenproporex in young and adult rats presented higher levels of damage index and frequency in the DNA. However, chronic administration of fenproporex in young and adult rats did not alter the levels of DNA damage in both parameters of comet assay. The present findings showed that acute administration of fenproporex promoted damage in DNA, in both young and adult rats. Our results are consistent with other reports which showed that other amphetamine-derived drugs also caused DNA damage. We suggest that the activation of an efficient DNA repair mechanism may occur after chronic exposition to fenproporex. Our results are consistent with other reports that showed some amphetamine-derived drugs also caused DNA damage.     

Effect of Acute and Chronic Administration of l-Tyrosine on Nerve Growth Factor Levels in Rat Brain

Most inborn errors of tyrosine catabolism produce hypertyrosinemia. Neurological manifestations are variable and some patients are developmentally normal, while others show different degrees of developmental retardation. Considering that current data do not eliminate the possibility that elevated levels of tyrosine and/or its derivatives may have noxious effects on central nervous system development in some patients, the present study evaluated nerve growth factor (NGF) levels in hippocampus, striatum and posterior cortex of young rats. In our acute protocol, Wistar rats (10 and 30 days old) were killed 1 h after a single intraperitoneal administration of l-tyrosine (500 mg/kg) or saline. Chronic administration consisted of l-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old); the rats were killed 12 h after the last injection. NGF levels were then evaluated. Our findings showed that acute administration of l-tyrosine decreased NGF levels in striatum of 10-day-old rats. In the 30-day-old rats, NGF levels were decreased in hippocampus and posterior cortex. On the other hand, chronic administration of l-tyrosine increased NGF levels in posterior cortex. Decreased NGF may impair growth, differentiation, survival and maintenance of neurons.     

Inhibition of mitochondrial respiratory chain in the brain of rats after hepatic failure induced by acetaminophen

To explore the effect of LYRM1 over-expression on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells, and to understand the underlying mechanisms, Rat myoblasts (L6) transfected with either an empty expression vector (pcDNA3.1Myc/His B) or a LYRM1 expression vector were differentiated into myotubes. Glucose uptake was determined by measuring 2-deoxy-D-[(3)H] glucose uptake into L6 myotubes. Western blotting was performed to assess the translocation of insulin-sensitive glucose transporter 4 (GLUT4). It was also used to measure the phosphorylation and total protein contents of insulin-signaling proteins, such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase (PI3K) p85, Akt, ERK1/2, P38, and JNK. LYRM1 over-expression in L6 myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1, PI3K (p85), and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, P38, and JNK. LYRM1 regulates the function of IRS-1, PI3K, and Akt, and decreases GLUT4 translocation and glucose uptake in response to insulin. These observations highlight the potential role of LYRM1 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity.     

Multimodal Retinal Vessel Analysis in CADASIL Patients

Purpose

To further elucidate retinal findings and retinal vessel changes in Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) patients by means of high resolution retinal imaging.

Methods

28 eyes of fourteen CADASIL patients and an equal number of control subjects underwent confocal scanning laser ophthalmoscopy (cSLO), spectral-domain optical coherence tomography (SD-OCT), retinal nerve fibre layer (RNFL) measurements, fluorescein and indocyanine angiography. Three vessel measurement techniques were applied: RNFL thickness, a semiautomatic software tool based on cSLO images and manual vessel outlining based on SD-OCT.

Results

Mean age of patients was 56.2±11.6 years. Arteriovenous nicking was present in 22 (78.6%) eyes and venous dilation in 24 (85.7%) eyes. Retinal volume and choroidal volume were 8.77±0.46 mm³ and 8.83±2.24 mm³. RNFL measurements showed a global increase of 105.2 µm (Control group: 98.4 µm; p = 0.015). Based on semi-automatic cSLO measurements, maximum diameters of arteries and veins were 102.5 µm (106.0 µm; p = 0.21) and 128.6 µm (124.4 µm; p = 0.27) respectively. Manual SD-OCT measurements revealed significantly increased mean arterial 138.7 µm (125.4 µm; p<0.001) and venous 160.0 µm (146.9; p = 0.003) outer diameters as well as mean arterial 27.4 µm (19.2 µm; p<0.001) and venous 18.3 µm (15.7 µm; p<0.001) wall thicknesses in CADASIL patients.

Conclusions

The findings reflect current knowledge on pathophysiologic changes in vessel morphology in CADASIL patients. SD-OCT may serve as a complementary tool to diagnose and follow-up patients suffering from cerebral small-vessel diseases.     

Efficient RNA interference in patients' acute lymphoblastic leukemia cells amplified as xenografts in mice

Background

Signaling studies in cell lines are hampered by non-physiological alterations obtained in vitro. Physiologic primary tumor cells from patients with leukemia require passaging through immune-compromised mice for amplification. The aim was to enable molecular work in patients' ALL cells by establishing siRNA transfection into cells amplified in mice.

Results

We established delivering siRNA into these cells without affecting cell viability. Knockdown of single or multiple genes reduced constitutive or induced protein expression accompanied by marked signaling alterations.

Conclusion

Our novel technique allows using patient-derived tumor cells instead of cell lines for signaling studies in leukemia.     

Mammalian metallothionein

Chemical, spectroscopic, and structural studies have established the metallothioneins (MTs) to be a widely occurring family of polypeptidic bioinorganic structures. They are distinguished by an extremely high metal (Zn, Cd, Cu) and Cys content and by the arrangement of these components in metal-thiolate clusters. By structural criteria the MTs have recently been subdivided into three classes (Fowler et al.,Experientia Suppl. 52, 19–22, 1987). Class I MTs include mammalian MTs and related forms. Class II MTs display no such relationships, and Class III MTs are atypical polypeptides made up of repetitive γ-glutamylcysteinyl units. Amino acid sequences of over 50 MTs are now known. In mammals, over 55% of the residues, including the 20 Cys, are conserved. Mammalian MTs are genetically polymorphous. Thus, in human tissues and cell lines closely related structures of ten functional isoMTs have been determined either by amino acid or nucleotide sequencing. A comparable degree of polymorphism also exists in the rabbit. Mammalian MTs have been inferred to bind a total of seven bivalent metal ions (Me) through thiolate coordination in two separate clusters, i.e., Me(II)₃(Cys)₉ and Me(II)₄(Cys)₁₁. This two-cluster model has now fully been confirmed by the spatial structures of rat MT-2 and rabbit MT-2a determined by 2D NMR spectroscopy in aqueous solution.