Opioid Peptide Gene Expression in the Primary Hereditary Cardiomyopathy of the Syrian Hamster I. REGULATION OF PRODYNORPHIN GENE EXPRESSION BY NUCLEAR PROTEIN KINASE C

Prodynorphin gene expression was investigated in adult ventricular myocytes isolated from normal (F1B) or cardiomyopathic (BIO 14.6) hamsters. Prodynorphin mRNA levels were higher in cardiomyopathic than in control myocytes and were stimulated by treatment of control cells with the protein kinase C (PKC) activator 1, 2-dioctanoyl-sn-glycerol. Both chelerythrine and calphostin C, two PKC inhibitors, abolished the stimulatory effect of the diglyceride and significantly reduced prodynorphin gene expression in cardiomyopathic myocytes. Nuclear run-off experiments indicated that the prodynorphin gene was regulated at the transcriptional level and that treatment of nuclei isolated from control cells with 1, 2-dioctanoyl-sn-glycerol increased prodynorphin gene transcription, whereas chelerythrine or calphostin C abolished this transcriptional effect. Direct exposure of nuclei isolated from cardiomyopathic myocytes to these inhibitors markedly down-regulated the rate of gene transcription. The expression of PKC-alpha, -delta, and -epsilon, as well as PKC activity, were increased in nuclei of cardiomyopathic myocytes compared with nuclei from control cells. The levels of both intracellular and secreted dynorphin B, a biologically active product of the gene, were higher in cardiomyopathic than in control cells and were stimulated or inhibited by cell treatment with 1,2-dioctanoyl-sn-glycerol or PKC inhibitors, respectively

Performance of two potentiometric fluoride determination methods in hard dental tissue

A comparison between two ion selective electrode (ISE) potentiometric methods is reported for determining the amount of fluoride in hard dental tissue after placement of fluoride-releasing dental restorations. The two methods are: (1) the direct method involving linear calibration (LC), and (2) a spiking method involving multiple standard additions (MA). Results showed that measurements performed by the LC method underestimate the amount of fluoride released by up to 30%. Recovery tests demonstrated that the use of MA and blank correction procedures is useful for an accurate and sensitive ISE determination of fluoride in hard dental tissues

Rescue of Impaired Angiogenesis in Spontaneously Hypertensive Rats by Intramuscular Human Tissue Kallikrein Gene Transfer

Angiogenesis represents a compensatory response targeted to preserve the integrity of tissues subjected to ischemia. The aim of the present study was to examine whether reparative angiogenesis is impaired in spontaneously hypertensive rats (SHR), as a function of progression of hypertension. In addition, the potential of gene therapy with human tissue kallikrein (HK) in revascularization was challenged in SHR and normotensive Wistar-Kyoto rats (WKY) that underwent excision of the left femoral artery. Expression of vascular endothelial growth factor and HK was upregulated in ischemic hindlimb of WKY but not of SHR. Capillary density was increased in ischemic adductor muscle of WKY (from 266±20 to 633±73 capillaries/mm 2 at 28 days, P <0.001), whereas it remained unchanged in SHR (from 276±20 to 354±48 capillaries/mm 2 , P =NS), thus compromising perfusion recovery as indicated by reduced plantar blood flow ratio (0.61±0.08 versus 0.92±0.07 in WKY at 28 days, P <0.05). In separate experiments, saline or 5×10 9 pfu adenovirus containing the HK gene (Ad.CMV-cHK) or the β-galactosidase gene (Ad.CMV-LacZ) was injected intramuscularly at 7 days after the induction of ischemia. Ad.CMV-cHK augmented capillary density and accelerated hemodynamic recovery in both strains, but these effects were more pronounced in SHR ( P <0.01). Our results indicate that native angiogenic response to ischemia is impaired in SHR, possibly as a result of defective modulation of endothelial cell mitogens. Supplementation with kallikrein, one of the growth factors found to be deficient in SHR, restores physiological angiogenic response utilitarian for tissue healing. Our discoveries may have important implications in vascular medicine for therapeutic benefit

Importance of Ezh2 polycomb protein in tumorigenesis process interfering with the pathway of growth suppressive key elements

An understanding of the mechanisms that uncover the dynamic changes in the distribution of the chromatin modifying enzymes and regulatory proteins on their target loci could provide further insight into the phenomenon of malignant transformation. Based on the current available data, it seems more and more clear that an abnormal expression of Ezh2, a member of the Polycomb group (PcG) protein, may be involved in the tumorigenesis process, in addition, different studies identify Ezh2 as a potential marker that distinguish aggressive prostate and breast cancer from indolent one. Recent investigation show that ectopic expression of Ezh2 provides proliferative advantage to primary cells through interaction with the pathways of key elements that control cell growth arrest and differentiation, like members of the retinoblastoma (Rb) family. Here, we outline how these pathways converge and we review the recent advances on the molecular mechanisms that promote cell cycle progression through deregulation of Ezh2 protein level, providing novel links between cancer progression and chromatin remodeling machineries

Thermal stability of lysozyme Langmuir-Schaefer films by FTIR spectroscopy

Fourier transform infrared spectroscopy has been applied to study the thermal stability of multilayer Langmuir- Schaefer (LS) films of lysozyme deposited on silicon substrates. The study has confirmed previous structural findings that the LS protein films have a high thermal stability that is extended in a lysozyme multilayer up to 200 °C. 2D infrared analysis has been used here to identify the correlated molecular species during thermal denaturation. Asynchronous 2D spectra have shown that the two components of water, fully and not fully hydrogen bonded, in the high-wavenumber range (2800-3600 cm-1) are negatively correlated with the amine stretching band at 3300 cm-1. On the grounds of the 2D spectra the FTIR spectra have been deconvoluted using three main components, two for water and one for the amine. This analysis has shown that, at the first drying stage, up to 100 °C, only the water that is not fully hydrogen bonded is removed. Moreover, the amine intensity band does not change up to 200 °C, the temperature at which the structural stability of the multilayer lysozyme films ceases

Activation and function of murine Cyclin T2A and Cyclin T2B during skeletal muscle differentiation

Cyclin-dependent kinase 9 (Cdk9) is a serine-threonine kinase, involved in many cellular processes. The regulatory units of Cdk9 are the T family Cyclins (T1, T2) and Cyclin K. Cyclin T2 has two forms termed Cyclin T2a and Cyclin T2b that arise by an alternative splicing of the primary transcript. Upon induction of muscle differentiation, MyoD recruits Cdk9/Cyclin T2 on muscle-specific gene promoter sequences. This complex is able to phosphorylate the C-terminal domain of RNA polymerase II, enhancing MyoD function and promoting myogenic differentiation. This work focuses on the characterization of two murine Cyclin T2 isoforms and the evaluation of the role of Cdk9/Cyclin T2 complexes during the skeletal muscle differentiation. This study emonstrated a predominant expression of isoform b in all stages of differentiation. Moreover, both isoforms of Cyclin T2 are able to activate the myogenic program but Cyclin T2b has a predominant role, in particular during the latest stages

Ramipril improves hemodynamic recovery but not microvascular response to ischemia in spontaneously hypertensive rats

Background Angiotensin converting enzyme (ACE) inhibition exerts positive effects on the microvasculature of normotensive animals, although this concept is not universally accepted. Recently, ACE inhibitors have been suggested to be useful for rescue in peripheral ischemia. Methods We investigated whether chronic treatment with the ACE inhibitor ramipril may have a positive impact on the defective healing response to ischemia that is typical of spontaneously hypertensive rats (SHR). Unilateral limb ischemia was induced in 20-week-old SHR by surgically removing the left femoral artery. Rats were allowed to regain consciousness and then were randomly allocated to treatment with ramipril (1 mg/kg body weight in drinking water) or vehicle for 28 days. Results The SHR failed to develop reparative angiogenesis in response to ischemia, thus having inadequate perfusion recovery. Ramipril reduced both tail-cuff systolic blood pressure (180 ± 7 v 207 ± 2 mm Hg in the vehicle group at 28 days, P &lt; .05) and intra-arterial mean blood pressure (115 ± 6 v 135 ± 5 mm Hg in the vehicle group, P &lt; .05). These effects were associated with increased responsiveness to endothelium-dependent vasodilatation by acetylcholine. Treatment with ramipril did not influence muscular capillary and arteriole density but accelerated the rate of perfusion recovery, leading to complete healing within 28 days after surgery. Conclusions These results indicate that ACE inhibition by ramipril may be useful for the treatment of peripheral vascular complications in hypertension

Renovascular hypertension in bradykinin B₂-receptor knockout mice

We evaluated whether kinins exert a protective action against the development of two-kidney, one clip (2K1C) hypertension, a model characterized by an activated renin-angiotensin system in the ischemic kidney and increased expression of the bradykinin (BK) B2 receptor in the contralateral kidney. BK B2-receptor knockout (B2 -/-), wild-type (B2 +/+), and heterozygous (B2 +/-) mice underwent clipping of the left renal artery, with the other kidney remaining untouched. Basal systolic blood pressure (SBP, via tail-cuff plethysmography) was higher in B2 -/-mice than in B2 +/- or B2 +/+ mice (121±2 versus 113±2 and 109±1 mm Hg; P&lt;0.05 for both comparisons). SBP did not change from basal values after sham operation, but it increased in mice that underwent clipping. The increase in SBP was greater in 2K1C B2 -/- mice than in B2 +/- or B2 +/+ mice (28±2 versus 14±2 and 14±2 mm Hg, respectively, at 2 weeks; P&lt;0.05 for both comparisons). Blockade of the BK B2 receptor by Icatibant enhanced the pressure response to clipping in B2 +/+ mice (29±2 mm Hg at 2 weeks). Intra-arterial mean blood pressure (MBP) was higher in 2K1C than in respective sham-operated mice, with the MBP difference being higher in B2 -/- mice (32 and 38 mm Hg, at 2 and 4 weeks, respectively), and higher in B2 +/+ mice given Icatibant (30 and 32 mm Hg) than in B2 +/+ mice without Icatibant (17 and 18 mm Hg). At 4 weeks, acute injection of an angiotensin type 1 receptor antagonist normalized the MBP of 2K1C hypertensive mice. A tachycardic response was observed 1 week after clipping in B2 -/- and B2 +/- mice, but this effect was delayed in B2 +/+ mice. However, the HR response to clipping in B2 +/+ mice was enhanced by Icatibant. Within each strain, heart weight to body weight ratio was greater in 2K1C hypertensive mice than in sham-operated control animals (B2 -/-: 5.7±0.1 versus 5.2±0.1; B2 +/+: 5.1±0.1 versus 4.5±0.1; P&lt;0.01 for both comparisons). The clipped kidney weight to nonclipped kidney weight ratio was consistently reduced in mice with 2K1C hypertension. Our results indicate that kinins acting on the BK B2 receptor exert a protective action against excessive blood pressure elevation during early phases of 2K1C hypertension

High-throughput CZE-UV determination of arginine and dimethylated arginines in human plasma

Experimental studies document that increased asymmetric dimethylarginine (ADMA) blood levels inhibit NOS significantly, reducing NO generation. ADMA measurement often needs sample cleanup by SPE prior to chromatography and precolumn derivatization that cannot be easily employed in a routine clinical setting. We set up a new reliable CE method to measure ADMA, symmetric dimethylarginine (SDMA), and arginine without sample extraction or precolumn derivatization in order to examine their concentrations in human plasma. Sample was concentrated prior to CE injection and analytes were monitored by UV detection. CE analysis was performed in an uncoated fused-silica capillary, 75 μm id and 60.2 cm length (50 cm to the detection window), injecting 1 s water plug (0.5 psi) followed by 10 s of the sample (0.5 psi). Separation was carried out in a 50 mmol/L Tris-phosphate run buffer at pH 2.30, 15°C and 15 kV (75 μA) at normal polarity. Recovery of plasma ADMA was 101-104% and inter-day CV was less than 3%. Assay performance was evaluated measuring the levels of arginine and its dimethyl derivatives in 77 subjects. Passing-Bablok regression and Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference CE-LIF assay are similar