Biomedical subjects

Barbara Rosati

Publications and source records attributed to Barbara Rosati.

8 recordsLinked to original sources

PubMed2025

Propionyl-CoA carboxylase subunit B regulates anti-tumor T cells in a pancreatic cancer mouse model.

Most human pancreatic ductal adenocarcinoma (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. Now, we show that implantation of Pik3ca-/- KPC (named αKO) cancer cells induces clonal enrichment of cytotoxic T cells infiltrating the pancreatic tumors. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression, and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with clonally enriched CD8+ T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated these clonally enriched T cells infiltrating p-αKO tumors, leading to slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers and this understanding may lead to improvement in immunotherapy for this difficult-to-treat cancer.

Animals↗

PubMed2007

Molecular basis of the T- and L-type Ca2+ currents in canine Purkinje fibres.

This study examines the molecular basis for the T-type and L-type Ca(2+) currents in canine Purkinje cells. The I(CaT) in Purkinje cells was completely suppressed by 200 nM kurtoxin, a specific blocker of both Ca(v)3.1 and Ca(v)3.2 channels. Since only Ca(v)3.2 mRNA is expressed at high levels in Purkinje fibres, being approximately 100-fold more abundant than either Ca(v)3.1 or Ca(v)3.3 mRNAs, it is concluded that the Ca(v)3.2 gene encodes the bulk of the T-type Ca(2+) channels in canine Purkinje cells. This conclusion is consistent with the sensitivity of the current to blockade by Ni(2+) ions (K(D) = 32 microM). For L-type channels, Ca(v)1.2 mRNA was most abundant in Purkinje fibres but a significant level of Ca(v)1.3 mRNA expression was also found. A comparison of the sensitivity to blockade by isradipine of the L-type currents in Purkinje cells and ventricular epicardial myocytes, which only express Ca(v)1.2, suggests that the Ca(v)1.3 channels make, at most, a minor contribution to the L-type current in canine Purkinje cells.

Animals↗

PubMed2006

Regional variation in mRNA transcript abundance within the ventricular wall.

Previous studies have shown that regional variation in ion channel gene expression contributes to electrical heterogeneity within the walls of the cardiac ventricles. To map the extent of regional variation in gene expression in the ventricular walls and to begin to understand its genesis we have performed a microarray analysis of gene expression in the epicardial and endocardial tissues of the rat adult left ventricle. While the vast majority of the genes are expressed at uniform levels across the ventricular wall, a total of 36 transcripts (representing less than 0.1% of the genes expressed in the ventricle) are expressed more abundantly in either epicardium or endocardium. One of these differentially expressed genes is the sodium channel gene Scn5a, which is expressed at higher levels in the endocardium than in the epicardium of rat heart. The transcription factor genes Irx3, Irx5 and Etv1 were found to be expressed in transmural gradients across the ventricular wall of rat heart and also of canine heart. The Irx3 and Irx5 genes were expressed in an inverse pattern to that of the Kcnd2 (Kv4.2) gene in rat heart, suggesting that these transcription factors may act as negative regulators of Kcnd2 expression in vivo.

Animals↗

PubMed2004

Regulation of ion channel expression.

A potentially important mechanism controlling ion channel expression is homeostatic regulation, which can act to maintain a stable electrophysiological phenotype in cardiac myocytes as well as to provide plasticity in response to genetic, pathological, or pharmacological insults. The capabilities and limitations of the homeostatic regulatory mechanisms that contribute to the control of cardiac ion channel expression are the primary topic of this review.

Animals↗

PubMed2004

Comparison of different probe-level analysis techniques for oligonucleotide microarrays.

Three different software packages for the probe-level analysis of high-density oligonucleotide microarray data were compared using an experiment-derived data set that was validated using real-time PCR. The efficiency with which these three programs could identify true positives in this data set was assessed. In addition, estimates of false-positive and false-negative rates were determined. The performance of the programs using very small data sets was also compared, and recommendations for use are suggested.

Animals↗

PubMed2003

Concordant expression of KChIP2 mRNA, protein and transient outward current throughout the canine ventricle.

Expression of the transient outward K+ current (Ito) was analysed in nine different regions of the canine ventricle. In addition to the previously described transmural gradients in the right and left ventricular free walls, an inverted U-shaped pattern of Ito expression was observed in the interventricular septum. The mRNA and protein expression for the K+ channel beta subunit KChIP2 were examined in the same tissues. The KChIP2 protein levels closely matched mRNA expression in all regions of the ventricle and paralleled the density of Ito. The global pattern of gene expression in human epicardial and endocardial tissue was examined using microarrays. Only 0.1 % of the genes expressed in the human ventricle displayed the same expression pattern as the KChIP2 gene in left ventricle. It is unlikely, therefore, that the reported distribution of KChIP2 protein within the ventricle can be explained by a cross-reaction of the anti-KChIP2 antibody with a different protein. It is concluded that transcriptional regulation of the KChIP2 gene is a primary determinant of Ito expression in heart.

Animals↗

PubMed2002

ERG K+ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death?

The ether-a-go-go-related genes (erg) are expressed in tissues other than heart and brain, in which human erg (HERG) K+ channels are known to regulate the repolarization of heart action potentials and neuronal spike-frequency accommodation. We provide evidence that erg1 transcripts and ERG proteins are present in rat chromaffin cells in which we could isolate a K+ current that was biophysically and pharmacologically similar to the ERG current. Firing frequency and catecholamine release were analyzed at the single-cell level by means of perforated patch-clamp and carbon fiber electrochemical detection. It was found that the blocking of ERG, KATP, and KCa channels led to hyperexcitability and an increase in catecholamine release. Combined immunocytochemical experiments with antibodies directed against phenylethanolamine N-methyltransferase and ERG channels suggested expression of these channels in epinephrine- but not in norepinephrine-containing cells. It is concluded that, in addition to being crucial in regulating the QT period in the heart, ERG channels play a role in modulating epinephrine, a fundamental neurotransmitter shaping cardiac function. This finding suggests that the sudden death phenotype associated with LQT2 syndrome mutations may be the result of an emotionally triggered increase in epinephrine in a long-QT running heart.

Animals↗

PubMed2002

Isolation of a long-lasting eag-related gene-type K+ current in MMQ lactotrophs and its accommodating role during slow firing and prolactin release.

Native rat lactotrophs express thyrotrophin-releasing hormone-dependent K+ currents consisting of fast and slow deactivating components that are both sensitive to the class III anti-arrhythmic drugs that block the eag-related gene (ERG) K+ current (I(ERG)). Here we describe in MMQ prolactin-releasing pituitary cells the isolation of the slowly deactivating long-lasting component (I(ERGS)), which, unlike the fast component (I(ERGF)), is insensitive to verapamil 2 microm but sensitive to a novel scorpion toxin (ErgTx-2) that hardly affects I(ERGF). The time constants of I(ERGS) activation, deactivation, and recovery from inactivation are more than one order of magnitude greater than in I(ERGF), and the voltage-dependent inactivation is left-shifted by approximately 25 mV. The very slow MMQ firing frequency (approximately 0.2 Hz) investigated in perforated patch is increased approximately four times by anti-arrhythmic agents, by ErgTx-2, and by the abrupt I(ERGS) deactivation. Prolactin secretion in the presence of anti-arrhythmics is three- to fourfold higher in comparison with controls. We provide evidence from I(ERGS) and I(ERGF) simulations in a firing model cell to indicate that only I(ERGS) has an accommodating role during the experimentally observed very slow firing. Thus, we suggest that I(ERGS) potently modulates both firing and prolactin release in lactotroph cells.

Action Potentials↗