Carbonic anhydrase in mammalian vascular smooth muscle.

Carbonic anhydrase (CA) is ubiquitously expressed and plays a pivotal role in acid-base balance, ion transport, and gas exchange. Limited observations by others, derived from functional, pharmacological, and histochemical studies, suggest that CA is present in vascular smooth muscle and is involved in vasoregulation. The present study, using measurements of bioactivity, inhibition characteristics, and immunohistochemical analysis, was undertaken to more fully evaluate CA in vascular smooth muscle. In isolated bovine aortic smooth muscle, which is devoid of erythrocytes, CA is present in low concentrations with a CO(2) hydration activity (at 0C) of 3.5 +/- 2.7 U/g. The I(50) for acetazolamide inhibition is 0.07 +/- 0.01 microM. Results with dorzolamide and bromopyruvate, selective inhibitors of the CA II and I isozymes, respectively, show that roughly 75% of the CA activity is accounted for by CA I, with 20% due to CA II. These results accord qualitatively with immunocytochemical staining with specific CA I and II antibodies, showing that both isozymes are present and that their staining co-localizes with cells positive for smooth muscle alpha-actin. These data establish the activity, inhibition, and isozyme pattern of carbonic anhydrase expression in mammalian vascular smooth muscle.     

Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

IntroductionAbnormal oxidative stress has been described in systemic sclerosis (SSc) and previous works from our laboratory demonstrated an increased generation of reactive oxygen species (ROS) by SSc fibroblasts and monocytes. This study investigated the ability of SSc T lymphocytes to produce ROS, the molecular pathway involved, and the biological effects of ROS on SSc phenotype.MethodsPeripheral blood T lymphocytes were isolated from serum of healthy controls or SSc patients by negative selection with magnetic beads and activated either with PMA or with magnetic beads coated with anti-CD3 and anti-CD28 antibodies. Intracellular ROS generation was measured using a DCFH-DA assay in a plate reader fluorimeter or by FACS analysis. CD69 expression and cytokine production were analyzed by FACS analysis. Protein expression was studied using immunoblotting techniques and mRNA levels were quantified by real-time PCR. Cell proliferation was carried out using a BrdU incorporation assay.ResultsPeripheral blood T lymphocytes from SSc patients showed an increased ROS production compared to T cells from healthy subjects. Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox. Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.ConclusionsSSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production. These data confirm lymphocytes as key cellular players in the pathogenesis of systemic sclerosis and suggest a crucial link between ROS and T cell activation.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0591-8) contains supplementary material, which is available to authorized users.     

Psychophysical measure of visual luminance contrast during a daily rhythm

ABSTRACT

This study evaluated visual sensitivity to luminance contrast during a daily period. Twenty-eight young male adults (M = 24.85; SD = 2.4) with normal color vision and 20/20 visual acuity participated in this study. The circadian pattern was assessed using the Karolinska Sleepiness Scale (KSS), the Pittsburgh Sleep Quality Index (PSQI), and a sleep diary. To measure the luminance contrast, we used version 11.0 of the Metropsis software with sine-element frequency stimuli for spatial frequencies of 0.2, 0.6, 1, 3.1, 6.1, 8.8, 13.2, and 15.6 cycles per degree of visual angle (cpd). The stimuli were presented on a 19-inch color cathode ray tube (CRT) video monitor with a resolution of 1024 × 786 pixels, an update rate of 100 Hz, and a photopic luminance of 39.6 cd/m2. There was a significant difference in KSS on the weekdays [χ2₍₂₎ = 20.27; p = .001] and in the luminance contrast for frequencies of 13.2 cpd [χ2₍₂₎ = 8.27; p = .001] and 15.6 cpd [χ2₍₂₎ = 13.72; p = .041]. The results showed greater stability of the measurement during the afternoon and a reduction in the visual sensitivity in the high spatial frequencies during the night.     

Inhibition of S/G2 phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.     

Mesenchymal/stromal gene expression signature relates to basal-like breast cancers, identifies bone metastasis and predicts resistance to therapies

Background

Mounting clinical and experimental evidence suggests that the shift of carcinomas towards a mesenchymal phenotype is a common paradigm for both resistance to therapy and tumor recurrence. However, the mesenchymalization of carcinomas has not yet entered clinical practice as a crucial diagnostic paradigm.

Methodology/Principal Findings

By integrating in silico and in vitro studies with our epithelial and mesenchymal tumor models, we compare herein crucial molecular pathways of previously described carcinoma-derived mesenchymal tumor cells (A17) with that of both carcinomas and other mesenchymal phenotypes, such as mesenchymal stem cells (MSCs), breast stroma, and various types of sarcomas. We identified three mesenchymal/stromal-signatures which A17 cells shares with MSCs and breast stroma. By using a recently developed computational approach with publicly available microarray data, we show that these signatures: 1) significantly relates to basal-like breast cancer subtypes; 2) significantly relates to bone metastasis; 3) are up-regulated after hormonal treatment; 4) predict resistance to neoadjuvant therapies.

Conclusions/Significance

Our results demonstrate that mesenchymalization is an intrinsic property of the most aggressive tumors and it relates to therapy resistance as well as bone metastasis.     

High prevalence of Cag-A positive H. pylori strains in ischemic stroke: a primary care multicenter study

Background: Previous studies suggested an association between CagA-positive H. pylori strains and ischemic stroke. The aim of the present study was to assess the prevalence of Helicobacter pylori infection and CagA status in patients with atherosclerotic stroke in the primary care setting.
Materials and methods: A total of 106 consecutive patients (age 76.6 ± 8 years; males 52%) with well-documented history of atherosclerotic stroke and 106 sex–age- (age 76.5 ± 9 years; males 52%) and social background-matched controls without relevant vascular diseases. Risk factors for ischemic stroke were recorded in all subjects. H. pylori infection was assessed by[13]C-urea breath test. A serologic assay for specific IgG against CagA was performed in infected subjects.
Results: A trend toward a higher prevalence of H. pylori was observed in cases (63%) with respect to controls (54%) without reaching a statistical significance. CagA positivity was associated to a higher risk of atherosclerotic stroke (adjusted odds ratio 2.69, 95% confidence interval 1.37–5.30).
Conclusions: Our findings suggest that CagA-positive strains of H. pylori are significantly associated to atherosclerotic stroke. This is not a merely confirmative study since it has been performed for the first time in the primary care setting and included only subjects with an active infection.     

The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.