Search

Malignant gliomas are lethal primary intracranial tumors. To date, little information on the role of deregulated genes in gliomas have been identified. As the involvement of miRNAs in the carcinogenesis is well known, we carried out a pilot study to identify, as potential biomarkers, differentially expressed microRNAs in blood samples of patients affected by glioma. We studied the miRNAs’ expression, by means of microarray and Real-Time PCR, in 30 blood samples from glioma patients and in 82 blood samples of patients suffering from: (a) various neurological disorders (n=30), (b) primary B-lymphoma of the Central Nervous System (PCNSL, n=36) and (c) secondary brain metastases (n=16). By quantitative real time reverse-transcriptase polymerase chain reaction (qRT-PCR), we identified significantly increased levels of two candidate biomarkers, miR-15b and miR-21, in blood of patients affected by gliomas. ROC analysis of miR-15b biomarker levels allowed to differentiate patients with tumour from patients without glioma. Furthermore, combined expression analyses of miR15b and miR-21 distinguished between patients with and without glioma (90% sensitivity and 100% specificity). In addition, a decrement in the expression levels of miR-16 characterized glioblastomas compared to low grade and anaplastic gliomas. In conclusion, this pilot study suggest that it’s possible to identify the disease state by meaning miR-15b and miR-21 markers in blood, while miR-16 can be used to distinguish glioblastoma from other grade gliomas. They can potentially be used as biomarkers for non-invasive diagnosis of gliomas; further studies are mandatory to confirm our preliminary findings.

The expression of aquaporins in the spermatozoa of the marine teleost gilthead sea bream (Sparus aurata) and their involvement in the motility activation process were investigated. Sperm motility was activated by a hyperosmotic shock, but it was completely inhibited by 10 μM HgCl2, such inhibition being partially recovered by beta-mercaptoethanol (ME). Conventional RT-PCR using primers specific for S. aurata aquaglyceroporin (glp) and aquaporin 1a (aqp1a) demonstrated the presence of both mRNAs in spermatozoa. Heterologous expression in Xenopus laevis oocytes showed that 10 and 100 μM HgCl2equally inhibited water and solute transport through S. aurata aquaporin 1a and S. aurata aquaglyceroporin, but treatment with ME only recovered aquaporin 1a-mediated water permeability. Western blot analysis using isoform-specific antisera on protein extracts from spermatozoa revealed bands that corresponded to the predicted molecular mass of S. aurata aquaglyceroporin (31 kDa) and S. aurata aquaporin 1a (28 kDa). The antisera also demonstrated that both aquaporins were localized in the head and flagellum of the spermatozoa. However, the immunoreaction at the plasma membrane of the spermatozoa head was more intense after the hyperosmotic activation, suggesting the translocation of both aquaporin 1a and aquaglyceroporin into the plasma membrane after the osmotic shock. This study therefore provides the first direct demonstration for the presence of aquaporins in fish sperm. The different sensitivities of S. aurata aquaporin 1a and S. aurata aquaglyceroporin to ME may explain the failure of this reducing agent to fully recover the mercurial inhibition of sperm motility, suggesting that these aquaporins may play different physiological roles during the activation and maintenance of sperm motility in sea bream.

Molecular mechanisms involved in sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus) have been studied. Our comparative study demonstrates that osmolality is the key signal in sperm motility activation in both species, whereas K+and Ca2+do not have any role. The straight-line velocity that resulted, however, was significantly different when measured in sperm activated with non-ionic and/or calcium-free solutions with respect to that measured in seawater-activated sperm. In both species, motility initiation depends on cAMP-dependent protein phosphorylation. The phosphorylation/dephosphorylation patterns that resulted in gilthead and striped sea bream were quite different. In gilthead sea bream, the phosphorylated proteins have molecular weights of 174, 147, 138, 70, and 9-15 kDa, whereas the dephosphorylated proteins have molecular weights of 76, 57, and 33 kDa. In striped sea bream, phosphorylation after sperm motility activation occurred on proteins of 174, 147, 103, 96, 61, 57, and 28 kDa, whereas only one protein of 70 kDa resulted from dephosphorylation. Matrix-assisted laser desorption ionization-time of flight analyses allowed identification of the following proteins: In gilthead sea bream, the 9-15 kDa proteins that were phosphorylated after motility activation include an A-kinase anchor protein (AKAP), an acetyl-coenzyme A synthetase, and a protein phosphatase inhibitor, and in striped sea bream, 103- and 61-kDa proteins that were phosphorylated after motility activation were identified as a phosphatase (myotubularin-related protein 1) and a kinase (DYRK3), respectively.

In PC Cl3 cells, a rat thyroid cell line, angiotensin (Ang II) activates the atypical protein kinase C-ζ (PKC-ζ) and the extracellular signal-regulated kinase (ERK) pathways. We here studied the Ang II effects on PC Cl3 cell proliferation. It was found that Ang II: (1) induced the phosphorylation of protein kinase B (PKB), (2) induced the growth-related early gene c-fos expression, (3) enhanced the cyclin E and p27kipexpression, (4) had no effects on Cdk2, and (5) did not affect the transition from G0/G1 to S phase. Inhibition of phosphoinositide-3kinase by LY294002 further increased the effect of Ang II on p27kipinduction, whilst PKCs inhibition by GF109203X decreased such effect. The role of PKC-ζ was recognized by the use of a synthetic myristoylated peptide with sequences based on the endogenous PKC-ζ pseudosubstrate and by PKC-ζ downregulation using the small interfering RNA (siRNA). Insulin had a replicating effect on PC Cl3 cells, induced the phosphorylation of PKB, decreased p27kipexpression and had no effect on the PKC-ζ cytosol-to-membrane translocation. PC Cl3 cell proliferation was induced more potently by simultaneous stimulation with insulin and Ang II than by stimulation with insulin alone, and the effect on p27kipexpression was similar to that obtained with insulin only. These observations demonstrate that in PC Cl3 cells Ang II causes a block in G1 phase, although both ERK and PKB pathways are activated, and this effect may be due to the upregulation of p27kipand PKC-ζ operativity.

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]ilevel and increased the peak of Ca2+entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+and Ba2+uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+exchanger was not implicated, since the rate of decrement to the basal [Ca2+]ilevel was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]ilevel after P2Y2stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+transients caused by P2Y2stimulation.

Solute carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene (pept2) structure, and determined mRNA tissue distribution. An expressed sequence tag (EST) cDNA (Integrated Molecular Analysis of Gene Expression; IMAGE) corresponding to zebrafish pept2was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3,238-bp functional clone. The complete open reading frame (ORF) was 2,160 bp and encoded a 719-amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevisoocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter (K0.5for glycyl-l-glutamine ∼18 μM at −120 mV and pH 7.5). Zebrafish pept2gene was 19,435 kb, thus being the shortest vertebrate pept2fully characterized so far. Also, zebrafish pept2exhibited 23 exons and 22 introns, whereas human and rodent pept2genes contain 22 exons and 21 introns only. Zebrafish pept2mRNA was mainly detected in brain, kidney, gut, and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homolog to the higher vertebrate inner ear, of the adult fish. Characterization of zebrafish pept2will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development.

Objective: The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents.Methods: We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration.Results: Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents.Conclusion: Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems.

In the present study we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the protein expression of sea bass sperm was affected by the cryopreservation procedure. The protein profiles differed between fresh and frozen-thawed semen as revealed by visual inspection and by image analysis software. We identified 163 spots in fresh sperm; among these, 13 were significantly decreased and 8 were absent in two-dimensional gel obtained with cryopreserved sperm. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in bio-informatics analysis (PeptIdent, Mascot, and MS-Fit). In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with amphibian G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524 of Brachidanio rerio. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins and among these, two could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.

The most common parameters used to evaluate sperm quality are motility rate and duration and fertilization ability. In this study, chemical and biochemical parameters of sea bass (Dicentrarchus labrax) sperm were investigated to find an alternative method for evaluating sperm fertilization ability before and after cryopreservation. The biochemical and chemical analyses were performed with fresh and frozen-thawed sperm and seminal plasma. To cryopreserve sperm, 250-μl straws were used. Fertilization ability was evaluated by inseminating eggs (obtained from hormonally stimulated females) with fresh and cryopreserved sperm. The results revealed a linear relationship (P< 0.05) between semen fertilization capacity and some seminal plasma (β-d-glucuronidase activity, potassium concentration) and sperm (ATP concentration, aspartate aminotransferase activity) parameters. Variations in semen fertilization rate could be best described by two multiple regression models: one including the sperm parameters and another including the seminal plasma parameters. For practical application, the use of simple regression models is of value. Fertilization rate in both fresh and cryopreserved sperm was reliably predicted by determining the ATP concentration or the β-d-glucuronidase activity or both.

This work reports recent data on mechanisms of cold adaptation exhibited by the Antarctic teleosts Trematomus bernacchii and Chionodraco hamatus. Analysis of fatty acid in intestinal mucosa brush border suggested that an increase in unsaturated fatty acid could be a mechanism for the preservation of cell membrane integrity and functionality. The investigation of several transporters involved in the regulation of cell homeostasis (Na+/K+-AT-Pase, Na+-D-glucose cotransport, Na+/H+ exchanger and Ca++-AT-Pase) showed kinetic characteristics that could explain part of the adaptation of these proteins to work at low temperature. The activity of carbonic anhydrase, involved in pH control both at intra-cellular and systemic level, was related to plasma buffer capacity.

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3'-diethylthiacarbocyanine iodide (DiS-C2(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking “in vivo” conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andKapp values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.

The bacteriophage T7 binary expression system is widely usedin vitrofor high level selective expression of cloned genes but its application toin vivomodels has not yet been investigated. In the present work, we show that coinjection into fertilized zebrafish eggs of pE1T7R, an expression plasmid bearing theT7 RNA polymerasegene driven by the cytomegalovirus (CMV) promoter, together with reporter vectors containing theEscherichia coli lacZgene driven by the T7 promoter, resulted in the efficient expression of the reporter gene in 24-h mosaic transgenic embryos. Conversely, embryos receiving an unrelated CMV-expression plasmid, instead of pE1T7R, lacked significant reporter gene activity, indicating the strict requirement of T7 polymerase to activate the T7 promoter in these embryos. The present study demonstrates the possibility of applying efficiently the bacteriophage T7 binary systemin vivoto a vertebrate model.

Summary: The p53 protein was identified in primary breast carcinomas by specific binding of PAb1801 and PAb240 antibodies. Using sodium dodecyl sulfate electrophoresis followed by immunoblotting on nitrocellulose membrane, the p53 protein was identified in 36 nuclear fractions obtained from 60 primary breast cancers; semiquantitation of p53 was performed by densitometric scanning. The total cathepsin D content, the estorgen and progesterone receptor concentration values and the axillary lymph node involvement were also assessed. Tumors expressing p53 had significantly higher levels of cathepsin D than those in which p53 was undetectable. p53 expression was strongly associated with low or negative estrogen receptor values; progesterone receptor concentrations were also significantly higher in p53-negative tumors than in those tumors with detectable p53 levels. Finally, a significant relationship between p53 expression and lymph node metastasis was observed. It was concluded that a positive association between p53 and cathepsin D values exists which is of prognostic interest in that both cathepsin D and p53 are associated with a high tumor grade and metastatic activity.

Summary: Seventy-three primary human breast cancers were analyzed to assess the presence of estrogen and progesterone receptors, the p29 protein, and the total cathepsin D status. No significant relationship was found between cathepsin D concentration and the presence of ER or PR, either by Fisher's exact test or Spearman's rank correlation (P>0.1). However, a significant association was found between cathepsin D and p29 (Fisher's exact test,P<0.001) and between cathepsin D and steroid receptor status in samples expressing both estrogen and progesterone receptors (positive by steroid binding assay and enzyme immunoassay) (P<0.05). This asociation was more significant in tissues expressing estrogen and progesterone receptors as well as p29 (P<0.001). In contrast, cathepsin D synthesis was not related to tumor size, lymph node involvement, or patient's age (P>0.05). Steroid receptors and cathepsin D were also assayed in samples of non-malignant tissue from 16 mastectomies; there was a significantly higher relative concentration of cathepsin D in the malignant specimens (Stundent'st-test,P<0.001).

Two types of gliomatosis cerebri exist: Type I and Type II. We report the results of a histological and genetic study of two cases of gliomatosis cerebri Type II, correlating these results with therapy and prognosis.