Salivary chromogranin A, but not α-amylase, correlates with cardiovascular parameters during high-intensity exercise.

INTRODUCTION: Several studies have shown that activation of the sympathetic nervous system results in the increased secretion of α-amylase (sAA), an enzyme produced by salivary glands. Recently, chromogranin A (CgA), a soluble protein costored and coreleased with catecholamines from the adrenal medulla and sympathetic nerve endings, has been proposed as a marker of sympathoadrenal medullary system (SAM) activity. The aim of this study was to investigate the behaviour of salivary chromogranin A (sCgA) and sAA during high-intensity exercise and to analyse their possible correlation with cardiovascular and psychological parameters. METHODS: Before and during a standardized treadmill stress test, and at 5, 15 and 30 min during the recovery phase, sCgA and sAA were monitored in 21 healthy men. The double product (DP) of blood pressure and heart rate responses, and the product of the subjective ratings of perceived exertion recorded at the final step (RPE) and the exercise duration were used as indices of cardiovascular and exercise intensity, respectively. RESULTS: With respect to baseline, significant (P < 0·001) increases in peak sCgA (median 64%) and sAA (median 86%) were observed at the end of exercise. During the recovery phase, sAA levels fell abruptly, whereas sCgA remained elevated (P < 0·001). Significant correlations emerged only for sCgA with respect to %DP (r = 0·84; P < 0·001) and last step-RPE (r = 0·82; P = 0·024). CONCLUSIONS: These data suggest sCgA as a reliable marker of SAM activation. Furthermore, the relationship between sCgA and exercise intensity highlights the potential use of this noninvasive parameter in monitoring the adrenergic response during intense physical stress.

Parallel regulation of PKC-alpha and PKC-delta characterizes the occurrence of erythroid differentiation from human primary hematopoietic progenitors.

OBJECTIVE: Erythroid differentiation is a process characterized by modulation of different proteins including phosphoinositide-related enzymes such as protein kinase C (PKC) isoforms. Because in different cell lines PKC-alpha and PKC-delta have been reported to be involved in the mechanisms controlling proliferation and differentiation, the aim of this study was to examine the relative involvement of these PKC isoforms in the development of CD235a+ erythroid cells from human healthy hematopoietic progenitors. MATERIALS AND METHODS: Erythroid differentiation from human primary hematopoietic progenitor cells was achieved by adopting the human erythroblasts mass amplification culture. Expression and activity of PKC isoforms and their relationship with proliferation and differentiation were investigated by morphologic analysis, reverse-transcriptase polymerase chain reaction, Western blotting, multiparametric flow cytometry, and transfection experiments. RESULTS: PKC-alpha was found expressed and phosphorylated in cells undergoing both proliferation and differentiation, although PKC-delta, largely expressed and activated during proliferation, was evidently downregulated during differentiation. Overexpression of PKC-delta-CAT scarcely influenced the development of glycophorin-A (CD235a)+ erythroid cells from hematopoietic progenitors, although overexpression of PKC-alpha-CAT strongly induced the development of CD235a+ erythroid cells. On the other hand, in PKC-alpha-CAT-transfected cells, pharmacologic inhibition of PKC-delta further increased the number of CD235a+ cells, although inhibition of PKC-alpha resulted in an evident impairment of the development of CD235a+ erythroid cells. CONCLUSIONS: Our results indicate that the suppression or at least a strong downregulation of PKC-delta, concomitant to PKC-alpha expression and activity, might be a cofactor to be further investigated and might be involved in the events regulating erythropoietin-induced erythroid differentiation from human primary hematopoietic progenitor cells.

A flow cytometry procedure for simultaneous characterization of cell DNA content and expression of intracellular protein kinase C-zeta.

A selective involvement of protein kinase C-zeta (PKC-zeta) in the events regulating cell proliferation has been recently proposed. Here we report a flow cytometric method allowing the simultaneous association of intracellular PKC-zeta expression or phosphorylation with each cell cycle phase. Current methods for flow cytometry analysis were applied to several cell lines and compared to the method developed in our laboratory. The latter includes 2% paraformaldehyde (PFA), as fixing agent, a permeabilization/saturation step by means of a solution containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, 0.05% NP-40, 0.25% lambda-carrageenan and 0.02% NaN3, followed by labelling with a primary antibody (PKC-zeta or P-PKC-zeta) and with the appropriate FITC-conjugated secondary antibody. Cells processed by such a method disclosed no substantial modification of light scattering features with respect to live cells. In addition, stainability with anti-PKC-zeta or anti-P-PKC-zeta antibodies was well preserved while stoichiometric staining of DNA with PI enabled accurate cell cycle analysis. Results show that a distinct up-regulation of P-PKC-zeta in G2/M phase occurs. The method here described, therefore, represents a simple, reproducible and conservative assay for a simultaneous assessment of intracellular PKC or P-PKC modulations within each cell cycle phase.

Functional mitral regurgitation: from normal to pathological anatomy of mitral valve.

Mitral valve (MV) is composed of several structures working in synchrony to open during diastole and close in systole within the high-pressure systemic environment. Its morphological features ensure a normal leaflet closure that prevents regurgitation of blood back into the left atrium causing loss of ventricular pressure and forward flow. The complex interactions of the normal MV are reliant on each component playing a complete role for the efficient working of the valve. In this review we firstly discuss the overall MV structure in terms of a complex make up of the annulus, the leaflets, their tendinous cords, and the supporting papillary muscles, and then the anatomical changes of each MV components due to left ventricular geometry and function alterations, underlying functional mitral regurgitation.

Walking training affects dehydroepiandrosterone sulfate and inflammation independent of changes in spontaneous physical activity.

OBJECTIVE: We hypothesized that physical exercise in postmenopausal women could interfere with the molecular interrelationship of the immune-endocrine system and be effective even in women in whom training determined a reduction of spontaneous physical activity (SPA). For this reason, we investigated the effects of an aerobic program on plasma dehydroepiandrosterone sulfate (DHEA-S) and cytokine levels in relationship to SPA modification. METHODS: Thirty-two postmenopausal women (mean [SD] age, 56.38 [4.33] y) were enrolled in the study. Inclusion criteria were as follows: age younger than 65 years, body mass index higher than 18.5 and lower than 35 kg/m2, no pharmacological treatments, and no history of chronic, cardiovascular, or orthopedic diseases. Before and after 3 months of walking training at moderate intensity (40-50 min, 4 d/wk), they were evaluated for SPA, body composition, energy intake, and levels of plasma cytokines (tumor necrosis factor α [TNF-α], interleukin [IL]-1α, IL-1ß, IL-2, IL-8, and IL-10), C-reactive protein, DHEA-S, cortisol, and estrogen. RESULTS: At baseline, SPA did not correlate with either DHEA-S level or cytokine levels. There was negative correlation between DHEA-S and both TNF-α and IL-2. After the intervention program, 16 women showed increased SPA, and 16 women showed decreased SPA. Independent of these changes in SPA, both TNF-α levels and cortisol-to-DHEA-S ratio decreased, whereas DHEA-S levels increased. CONCLUSIONS: In postmenopausal women, walking training, rather than SPA, influences DHEA-S and cytokine concentrations and their correlations, thus interfering with adrenal steroids and the inflammatory markers network. Physical exercise acts in parallel on menopausal neuroendocrine alterations and on the systemic inflammatory profile independent of SPA changes.

Enhancement of TRAIL cytotoxicity by AG-490 in human ALL cells is characterized by downregulation of cIAP-1 and cIAP-2 through inhibition of Jak2/Stat3.

The ability of death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to selectively kill a variety of cancer cells has been largely described, but one of the major concerns with the treatment is the occurrence of drug resistance and possible toxic side effects. Here, we report that TRAIL induces apoptosis in Jurkat and SUPT1 T cell lines and in human T-ALL blasts but not in healthy subject-derived peripheral blood mononuclear cells. In parallel, the treatment with TRAIL and Tyrphostin (AG-490), a selective Janus kinase 2 inhibitor, produces an evident enhancement of cytotoxicity, characterized by a significant inhibition of Stat3 phosphorylation compared to controls or to TRAIL alone-treated samples, and associated with a dramatic decrease of both cIAP-1 and cIAP-2 mRNA levels. Downregulation of cIAP-1 and cIAP-2 by specific small interference RNAs significantly amplifies TRAIL-reduced cytotoxicity. All together, these findings strongly indicate that cIAP-1 and cIAP-2 downregulation is a fundamental step in the signaling pathways mediating the combinatorial effect of TRAIL and AG-490 on T cell leukemia. These findings may help to open new routes for the development of less toxic pharmacological strategies in the treatment of patients affected by TRAIL-sensitive leukemias.

Abeta(1-42) stimulated T cells express P-PKC-delta and P-PKC-zeta in Alzheimer disease.

The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction, and involvement of some PKC isoforms in T-cell activation has been demonstrated. Nevertheless, very little is known about their involvement in the Amyloid beta (Abeta)-dependent molecular signals in the T lymphocytes of Alzheimer disease (AD) patients. Therefore, the aim of this study was to investigate the involvement of PKC-alpha, PKC-delta and PKC-zeta expression and activity in the signaling machinery activated in Abeta-reactive T cells, in adult healthy individuals, elderly healthy subjects, and from patients with AD. The results show that in peripheral T-cells from early AD patients, Abeta(1-42) produced a distinct subpopulation highly expressing P-PKC-delta, while in severe AD patients the same treatment induced two distinct P-PKC-delta and P-PKC-zeta T-cell subpopulations. Such subpopulations were not noticeable following CD3/CD28 treatment of the same samples or after treatment of peripheral T cells from healthy adult or elderly subjects with Abeta(1-42) or with CD3/CD28. We believe that these findings may be of help in possible attempts to develop further diagnostic strategies useful for the characterization of AD.

Erythroid cell differentiation is characterized by nuclear matrix localization and phosphorylation of protein kinases C (PKC) alpha, delta, and zeta.

Protein kinases C (PKC) zeta expression and phosphorylation at nuclear level during dimethyl sulfoxide (DMSO)-induced differentiation in Friend erythroleukemia cells have been previously reported, suggesting a possible role of this PKC isoform in the DMSO-related signaling. In order to shed more light on this tantalizing topic, we investigated PKC intracellular and sub-cellular localization and activity during DMSO-induced erythroid differentiation. Results indicated that at least PKC alpha, zeta, and delta are strongly and temporally involved in the DMSO-induced differentiation signals since their expression and phosphorylation, though at different extents, were observed during treatments. Intriguingly, while PKC alpha and zeta associate to the nuclear matrix during the differentiation event, PKC delta appears to be residentially associated to the nuclear matrix. Furthermore, an evident downregulation of the beta-globin gene transcription (differentiation hallmark) was detected upon a progressive inhibition of these PKC isoforms by means of specific inhibitors, indicating, therefore, that PKC alpha, zeta, and delta phosphorylation play a crucial role in the control of erythroid differentiation.