Flow cytometric analysis of protein phosphorylation in the hematopoetic system

Cellular growth and differentiation in blood cells are regulated by the phosphorylation status of growth factor receptors and downstream proteins. Protein kinases and phosphatases balance the homeostasis of protein phosphorylation. Various diseases are associated with alterations in these tightly regulated processes. Aberrations have been proved to be of diagnostic value and might enhance the pathophysiological insight into the origin of the disease. However, quantitation of protein phosphorylation is currently not feasible in a clinical situation. We developed a flow cytometric methodology which enables for direct investigation of protein phosphorylation in cell populations defined by multi-color flow cytometry. This assay does not only overcome drawbacks of traditional methodologies (e.g. Western blotting) but also allows quantitative analyses even in rare cell populations. We accurately examined phosphorylation levels in different cell populations of hematological interest and especially analyzed CD34+ hematopoietic progenitor cells. CD34+ cells in bone marrow and in cord blood contained similar, low levels of phosphotyrosine. Circulating pheripheral blood system cells PBSC in patients exposed to G-CSF for stem cell mobilization exhibited significantly increased levels of phosphotyrosine. In vitro exposure of CD34+ progenitors to growth factors (G-CSF, IL-3, SCF) raised the levels of tyrosine phosphorylation in bone marrow and cord blood. Effects were dose and time dependent. Interestingly, in vivo stimulated CD34+ PBSC could not be further stimulated in vitro. In conclusion, we present a new powerful methodology for analysis of protein phosphorylation in hematological specimens. The method does not only allow for accurate detection of phosphorylation levels in vivo, but also enables for quantitative analysis of growth factor receptor stimulation in vitro and in vivo.     

Blocking of protein pancreatic secretion by maximal secretion infusion in the dog

In conscious chronic gastric and pancreatic fistula dogs (Thomas cannula), secretin was perfused for three hours with a submaximal (GIH, 1.0 C.U./kg.) and a maximal dose (GIH, 8.0 c.u./kg.), according to the following schedule: 1. First hour submaximal stimulus; 2. second hour maximal stimulus; 3. third hour submaximal stimulus. The alkaline and protein components of pancreatic secretion were analyzed in 20-minute sample collections thoughout the three hours. The same protocol was followed in anesthetized dogs subjected to a mind line laparotomy. A biopsy of the pancreatic gland was taken before (control) and at the end of each perfused dose. The secretion showed a significant increase of protein concentration and output when passing from the maximal to the last submaximal secretin perfusion dose. These findings correlated well with the piling up of zymogen and prozymogen granules in the apical zone of the acinar cells during maximal secretin perfusion, with their subsequent discharge into the acinar lumen upon abrupt reversal to the initial secretin submaximal dose. The study confirms that secretin influences pancreatic protein secretion and indicates in addition, that pharmacologic doses of the hormone, have the capacity to block acinar cell zymogen granule release.     

Effect of chlorine-containing pesticides on the hemolytic stability and acetylcholinesterase activity of erythrocytes

The hemolytic action of the chlorine-containing pesticides, trichloroacetic acid (TCA), pentachlorophenolate (PCP), chlorophos and pentachloronitrobenzene (PCNB) on red cells was studied, as were their effects on acetylcholinesterase activity and resistance of red cells to mechanical hemolysis by ultrasound. The modifying action of pesticides on red cell membranes was shown to lead to their mechanical resistance. TCA, PCP and chlorophos were found to sensitize red cells whereas PCNB to make them resistant to the mechanical action of ultrasound. The kinetic characteristics of the structural functional disorders of red cells might be used as quantitative criteria of the efficacy of the action of pesticides on the cells.     

Evidence for soluble and nuclear site I binding of estrogens in human aorta

The purpose of this study was to establish the estrogen receptor (ER) expression and content in human aorta fragments removed at the time of by-pass surgery. To this end, we adopted a radioligand binding assay to evaluate either soluble (S) or nuclear (N) ER using dextran-coated charcoal (DCC) and filtration methods, respectively. To better define the intratissular distribution and content of ER, we also measured the presence of a 27 kDa heat shock protein (HSP27), a well established ER-associated protein, using D5 monoclonal antibody. Finally, we analysed the different molecular isoforms of both S and N ER using size exclusion-high performance liquid chromatography (SE-HPLC). High affinity (type I) sites of estrogen binding were detected in 17 out of 19 samples in either S or N fraction, although only 9 out of 19 cases displayed site 1 ER in both cell compartments. ER levels in aortic tissues, detected by radioligand method, compare well with those we have found in other hormone-sensitive human cancer tissues and cells. SE-HPLC analysis revealed two main receptor isoforms in the soluble fraction, having 65 kDa and 18 kDa molecular mass, while a minor component of 29 kDa was also found; the nuclear fraction displayed again two major components of 38 and 23 kDa. Using the HSP27 immunohistochemistry we observed a major staining occurring in smooth muscle cells (SMC), with an increasing intensity towards the lumen. All samples, including the ER negative ones, exhibited some degree of histochemical staining. Using an arbitrary cut-off value, 7 out of 12 samples displayed a highly positive staining, 6 of which showed nuclear ER. Furthermore, SE-HPLC separation indicated the presence of a 64.9 kDa component in the soluble fraction, according to the well known relative molecular mass of ER. Following HSP27 immunohistochemistry, the overall staining intensity in aortic SMC approaches that seen in endometrial and breast epithelia, whilst the muscle ER content is generally lower. Although our data are compatible with a direct role of estrogens in arterial function, the extent of the link with arterial disease remains to be established.     

Microprocessor-based portable system for heart condition diagnosis

Early diagnosis of heart disease is typically based on a cassette recording of the electrocardiogram (ECG) signal which is then studied and analysed using a microcomputer. The system is bulky, unreliable and prone to mechanical failure. This paper presents the design and implementation of a compact microprocessor-based portable system used for heart condition diagnosis over a long period. The system reads, stores and analyses the ECG signals repetitively in real time for a specified period. The diagnostic data and samples of ECG signals are stored throughout the test period. The system hardware and software design are oriented towards a single-chip microcomputer-based system, hence minimizing size. The operating algorithm is based on a logical approach to ECG signal diagnosis and hence requires little memory.     

Function of the residual myocardium after infarct and prognostic significance

Changes of the left ventricle after myocardial infarction are characterized by geometric, structural, and vascular alterations, which have been summarized under the term "remodeling". This process takes place in the infarct region as well as in the surviving myocardium. Depending on to the size of infarction and the degree of neurohumoral activation, the left ventricle demonstrates diastolic dysfunction which may finally lead to systolic failure. The residual myocardium develops progressive myocyte hypertrophy and interstitial fibrosis. These structural alterations are due to changes in loading conditions and stimulation of the neurohumoral system with an activation of local paracrine and autocrine factors. Myocardial function can be assessed by different non-invasive (echocardiography, radionuclide ventriculography, magnetic resonance imaging, etc.) or invasive methods (e.g., simultaneous pressure-volume measurements). "Myocardial tagging" based on magnetic resonance imaging allows the assessment of 3D-motion of the left ventricle by labelling specific myocardial regions with a rectangular grid. A systolic "wringing" motion with clock-wise rotation at the base and counter-clockwise rotation at the apex has been described in normal subjects. In the ischemic myocardium, delayed relaxation with a prolonged back-rotation (untwisting) has been reported during early diastole, whereas decreased systolic contraction with delayed diastolic rotation has been observed in non-Q-wave infarction. In patients with anterolateral aneurysms, a complete loss of systolic rotation has been demonstrated. The prognostic significance of LV "remodeling" has been emphasized by several authors: The size of infarction, LV volume, LV ejection fraction, as well as the degree of neurohumoral activation have been identified as being associated with an unfavorable clinical outcome. Yearly mortality rates have been reported to range between 15 and 17% in patients with large infarcts and marked LV dilatation and between 3 and 7% in patients with small to medium-sized infarcts.     

Effect of inflammation on the proliferation of human gingival epithelial cells in vitro

The adaptive or pathologic responses of epithelial cells to inflammation are poorly characterized. The purpose of this study was to determine if epithelial cells cultured from clinically healthy and inflamed human gingival tissues express differences in proliferation rate and viability. Briefly, the inflammation status of individual donor sites from 101 patients was visually assessed at the time of periodontal surgery and categorized as either non-to-slightly inflamed, moderately inflamed, or severely inflamed. Discarded gingival tissues were then processed to obtain primary cell cultures, for which proliferation rates were determined by calculating the ratio of mean population doublings to the number of days required for cultures to become confluent. In general, the cells in the minimally inflamed group exhibited characteristics different than cells in the moderately and severely inflamed groups. Specifically, the cells obtained from clinical sites which exhibited no-to-slight inflammation had a significantly higher mean proliferation rate than cells in either the moderate inflammation group or the severe inflammation group. Based on trypan blue exclusion, the cells obtained from clinical sites which exhibited no-to-slight inflammation also were more viable than cells obtained from sites with moderate inflammation or severe inflammation. Microscopic evaluation showed morphological changes associated with increased inflammation. Cell cycle analysis by fluorescent-activated cell sorting (FACS) revealed a directly proportional relationship between the degree of inflammation and apoptosis, and a strong inversely proportional trend between the degree of inflammation and the numbers of cells undergoing mitosis. Taken together, these data suggest that epithelial cell proliferation and viability are inversely associated with the degree of gingival inflammation, once a putative "adaptive threshold" is exceeded. Elucidation of the underlying mechanisms will likely lead to improvements in clinical diagnosis and treatment.     

Sotahexal pharmacodynamics in patients with ischemic heart disease

Therapeutic and prophylactic antiarrhythmic efficacy of sotalol hydrochloride (Sotahexal, "Hexal", Germany) and its effects in intracardiac hemodynamics and ECG parameters were evaluated in 95 patients with ischemic heart disease (IHD). The highest response to the drug was observed in ventricular extrasystoles, arterial flutter and fibrillation. Acute episodes of arrhythmia are managed by bolus administration of Sotahexal [correction of Hexal]. In this case greater risk of side effects exists. It is desirable to decide on the drug dose, mode of administration on the individual basis with due consideration of the risks and dangers which could be avoided in case of adequate instrumental control.