Heterogeneous expression of phenobarbital-inducible cytochrome P-450 genes within the hepatic acinus in the rat

Within the hepatic acinus, the functional unit of liver parenchyma, the induction of cytochrome P-450 protein by phenobarbital is manifested primarily in hepatocytes located closer to the hepatic venule, i.e., distal hepatocytes. The objective of this study was to determine the levels of cytochromes P-450b and P-450e mRNAs in populations of hepatocytes originating in the proximal or distal half of the liver acinus in the rat, as an approach to the elucidation of the mechanisms responsible for the heterogeneous zonal expression of cytochrome P-450 protein. The development of a new method to isolate hepatocytes originating from the proximal or distal half of the liver acinus enabled the measurement of total cytochrome P-450 content and of cytochromes P-450b and P-450e mRNAs in these hepatocytes. Levels of cytochromes P-450b and P-450e mRNAs were assessed in proximal and distal hepatocytes by Northern blot hybridization of poly(A+)RNA with a cDNA recognizing sequences of these two cytochromes. The kinetics of induction were defined by measuring these parameters after a single phenobarbital injection. Cytochrome P-450 mRNA levels reached maximum induction at 16 hr, returning to basal values by 48 hr. In contrast, total cytochrome P-450 microsomal protein content reached maximum induction after 33 hr. Hepatocytes of the distal half of the hepatic acinus responded to phenobarbital with higher levels of cytochromes P-450b and P-450e mRNAs than proximal hepatocytes. These results indicated that there is modulation of the expression of the cytochromes P-450b and P-450e genes within the hepatic acinus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38328/1/1840060522_ftp.pd

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

Cytochrome p-450 gene expression in the functional units of the fetal liver

Hepatocytes of the right and left lobes of the fetal liver are surrounded by different microenvironments. The right and left lobes of the fetal liver are perfused by vascular systems carrying different concentrations of oxygen and constitute distinct functional units. The aim of this study was to assess the expression of the phenobarbital-inducible cytochrome P-450 b,e genes in hepatocytes of the right and left fetal liver lobes in mice. Northern-blot analysis using [ 32 P]cDNAs and quantitative dot-blot hybridization were performed to assess the size and levels of these mRNAs in the right and left fetal liver lobes. In fetal mice, the levels of cytochrome P-450, b,e mRNAs were higher in the left than in the right fetal liver lobe. During the last days of gestation and in the immediate postnatal period, the levels of liver cytochrome P-450 b,e mRNAs increased predominantly in the left liver lobe. In contrast, the levels of albumin and Α-fetoprotein mRNAs (genes studied to assess the specificity of these findings) were similar in each of functional units of the fetal liver. Phenobarbital induction of cytochromes P-450 b,e mRNAs was not observed in either of the fetal liver lobes. Postnatally, phenobarbital induced these cytochromes similarly in the right and left liver lobes. Therefore, the microenvironment surrounding fetal hepatocytes seems to influence the expression of the cytochrome P-450 b,e genes. This lobar heterogeneity of expression disappears as the pattern of adult liver circulation is attained.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38336/1/1840080222_ftp.pd

Phenobarbital induction of cytochrome p-450 b,e genes is dependent on protein synthesis

Phenobarbital induces liver cytochrome P-450 b,e proteins mainly by increasing the rate of transcription of these genes. The mechanism responsible for the phenobarbital increment in the rate of transcription of cytochrome P-450 b,e genes is unknown. The objective of this study was to assess whether active protein synthesis was needed for phenobarbital to induce the liver cytochrome P-450 b,e genes. Cycloheximide (2 mg per kg, i.p.) was administered 90 min prior to a single inductive dose of phenobarbital (80 mg per kg, i.p.) and mRNAS measured at 3, 6 and 12 hr by dot-blot hybridization. While phenobarbital increased cytochrome P-450 b,e mRNAs about 12-fold at 3 hr, this induction was abolished by cycloheximide. To define whether the absence of protein synthesis in hepatocytes inhibited the phenobarbital induction of cytochrome P-450 at the transcriptional level, in vitro transcription rates using isolated nuclei were measured. After phenobarbital administration, there was about a 20-fold increment in transcriptional rate of cytochrome P-450 b,e genes. This increment was abolished by prior injection of cycloheximide. It is proposed that either preexisting regulatory proteins or transacting factors dependent on active protein synthesis participate in the regulation of liver cytochrome P-450 b,e gene transcription after phenobarbital.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38337/1/1840080223_ftp.pd

HGF-Induced PKCζ Activation Increases Functional CXCR4 Expression in Human Breast Cancer Cells

The chemokine receptor CXCR4 and its ligand CXCL12 have been shown to mediate the metastasis of many malignant tumors including breast carcinoma. Interaction between hepatocyte growth factor (HGF) and the Met receptor tyrosine kinase mediates development and progression of cancers. HGF is able to induce CXCR4 expression and contributes to tumor cell invasiveness in breast carcinoma. However, the mechanism of the CXCR4 expression modulated by c-Met-HGF axis to enhance the metastatic behavior of breast cancer cells is still unclear. In this study, we found that HGF induced functional CXCR4 receptor expression in breast cancer cells. The effect of HGF was specifically mediated by PKCζ activity. After transfection with PKCζ-siRNA, the phosphorylation of PKCζ and CXCR4 was abrogated in breast cancer cells. Interference with the activation of Rac1, a downstream target of HGF, prevented the HGF-induced increase in PKCζ activity and CXCR4 levels. The HGF-induced, LY294002-sensitive translocation of PKCζ from cytosol to plasma membrane indicated that HGF was capable of activating PKCζ, probably via phosphoinositide (PI) 3-kinases. HGF treatment also increased MT1-MMP secretion. Inhibition of PKCζ, Rac-1 and phosphatidylinositol 3-kinase may attenuate MT1-MMP expression in cells exposed to HGF. Functional manifestation of the effects of HGF revealed an increased ability for migration, chemotaxis and metastasis in MDA-MB-436 cells in vitro and in vivo. Our findings thus provided evidence that the process of HGF-induced functional CXCR4 expression may involve PI 3-kinase and atypical PKCζ. Moreover, HGF may promote the invasiveness and metastasis of breast tumor xenografts in BALB/c-nu mice via the PKCζ-mediated pathway, while suppression of PKCζ by RNA interference may abrogate cancer cell spreading

Adjunctive Brivaracetam in Focal Epilepsy: Real-World Evidence from the BRIVAracetam add-on First Italian netwoRk STudy (BRIVAFIRST)

Background: In randomized controlled trials, add-on brivaracetam (BRV) reduced seizure frequency in patients with drug-resistant focal epilepsy. Studies performed in a naturalistic setting are a useful complement to characterize the drug profile. Objective: This multicentre study assessed the effectiveness and tolerability of adjunctive BRV in a large population of patients with focal epilepsy in the context of real-world clinical practice. Methods: The BRIVAFIRST (BRIVAracetam add-on First Italian netwoRk STudy) was a retrospective, multicentre study including adult patients prescribed adjunctive BRV. Patients with focal epilepsy and 12-month follow-up were considered. Main outcomes included the rates of seizure‐freedom, seizure response (≥ 50% reduction in baseline seizure frequency), and treatment discontinuation. The incidence of adverse events (AEs) was also considered. Analyses by levetiracetam (LEV) status and concomitant use of strong enzyme-inducing antiseizure medications (EiASMs) and sodium channel blockers (SCBs) were performed. Results: A total of 1029 patients with a median age of 45 years (33–56) was included. At 12 months, 169 (16.4%) patients were seizure-free and 383 (37.2%) were seizure responders. The rate of seizure freedom was 22.3% in LEV-naive patients, 7.1% in patients with prior LEV use and discontinuation due to insufficient efficacy, and 31.2% in patients with prior LEV use and discontinuation due to AEs (p < 0.001); the corresponding values for ≥ 50% seizure frequency reduction were 47.9%, 29.7%, and 42.8% (p < 0.001). There were no statistically significant differences in seizure freedom and seizure response rates by use of strong EiASMs. The rates of seizure freedom (20.0% vs. 16.6%; p = 0.341) and seizure response (39.7% vs. 26.9%; p = 0.006) were higher in patients receiving SCBs than those not receiving SCBs; 265 (25.8%) patients discontinued BRV. AEs were reported by 30.1% of patients, and were less common in patients treated with BRV and concomitant SCBs than those not treated with SCBs (28.9% vs. 39.8%; p = 0.017). Conclusion: The BRIVAFIRST provided real-world evidence on the effectiveness of BRV in patients with focal epilepsy irrespective of LEV history and concomitant ASMs, and suggested favourable therapeutic combinations

Sustained seizure freedom with adjunctive brivaracetam in patients with focal onset seizures

The maintenance of seizure control over time is a clinical priority in patients with epilepsy. The aim of this study was to assess the sustained seizure frequency reduction with adjunctive brivaracetam (BRV) in real-world practice. Patients with focal epilepsy prescribed add-on BRV were identified. Study outcomes included sustained seizure freedom and sustained seizure response, defined as a 100% and a ≥50% reduction in baseline seizure frequency that continued without interruption and without BRV withdrawal through the 12-month follow-up. Nine hundred ninety-four patients with a median age of 45 (interquartile range = 32–56) years were included. During the 1-year study period, sustained seizure freedom was achieved by 142 (14.3%) patients, of whom 72 (50.7%) were seizure-free from Day 1 of BRV treatment. Sustained seizure freedom was maintained for ≥6, ≥9, and 12 months by 14.3%, 11.9%, and 7.2% of patients from the study cohort. Sustained seizure response was reached by 383 (38.5%) patients; 236 of 383 (61.6%) achieved sustained ≥50% reduction in seizure frequency by Day 1, 94 of 383 (24.5%) by Month 4, and 53 of 383 (13.8%) by Month 7 up to Month 12. Adjunctive BRV was associated with sustained seizure frequency reduction from the first day of treatment in a subset of patients with uncontrolled focal epilepsy

Spatial Memory of Heterozygous Staggerer (Rora(+)/Rora(sg)) Versus Normal (Rora(+)/Rora(+)) Mice During Aging.

Heterozygous staggerer mice (Rora+/Rorasg) and control mice (Rora+/Rora+) of the same C57BL/6J strain background were tested in a spontaneous alternation task at 3 to 24 months old. The results demonstrated a decrement in long-term working memory as early as 6 months in Rora+/Rora+ mice and at 3 months in Rora+/Rorasg mice. Previous studies showed that in both cases, neuronal number in the cerebellar cortex was normal (Doulazmi et al. [1999]). This suggests that age-dependent decrease in long-term working memory would be due to fine structural or biochemical changes preceding neuronal death in the cerebellum. Such subtle changes would occur more precociously in Rora+/Rorasg than in Rora+/Rora+ mice. Also, short-term working memory was preserved in Rora+/Rora+ mice as old as 24 months, but was impaired in 6-month-old Rora+/Rorasg mice