Ave Minajeva

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca2+ was induced by 5 mM caffeine and the peak of relative tension (T/Tmax) and the area under isometric tension curves, ST, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca2+] in the fibres during the release was estimated using steady state [Ca2+]/tension relationship. Peak [Ca2+] and integral of free Ca2+ transients (S[Ca2+]f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg2+ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/Tmax was only 40% and S[Ca2+]f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both ST and S[Ca2+]f were more sensitive to the loading conditions than T/Tmax and peak [Ca2+]. The data suggest that Ca2+ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK.

Sarcoplasmic reticulum function in determining atrioventricular contractile differences in rat heart

The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+dT/d t) and relaxation (-dT/d t) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas +dT/d t and -dT/d t(normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca2+-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the α-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+ dT/dt) and relaxation (-dT/dt) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas + dT/dt and -dT/dt (normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+ concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+ adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca(2+)-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the alpha-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.

Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles

The present study was undertaken to compare the effects of hypothyroidism and hyperthyroidism on sarcoplasmic reticulum (SR) Ca2+-pump activity, together with assessment of the functional role of SR in providing activator Ca2+ under these altered thyroid states. In response to a shift from hypothyroid to hyperthyroid state, a 10 fold and 2 fold increase in SR Ca2+-pump activity in atria and ventricles, respectively, were observed. This was associated with the 8-9 fold increases in atrial contractility (+dT/dt) and relaxation (-dT/dt), but only with a 3-4 fold increase in their ventricular counterparts. Also, the recirculation fraction of activator Ca2+ (RFA) increased to a far greater extent in atria (4 fold) than in papillary muscles, and the relative increment in inhibition of developed tension by ryanodine became 3 times larger in atria than in papillary muscles. A positive force-frequency relationship (FFR) was observed in hypothyroid atria, whereas the hyperthyroid atria, hypothyroid and hyperthyroid papillary muscles showed a negative FFR. These results suggest the greater role of transsarcolemmal (SL) Ca2+ and smaller role of SR Ca2+ in activating contraction in hypothyroid atria compared to other preparations. Thyroid hormones decrease the contribution of SL and increase that of SR in providing activator Ca2+ to the greater extent in atria than in ventricles. This effect of thyroid hormones is based on larger stimulation of SR Ca2+-pump in atria compared to ventricles.

Relationship between neovascularization and degenerative changes in herniated lumbar intervertebral discs.

PurposeLumbar disc degeneration may be associated with intensity of neovascularization in disc herniations. Our study was designed to evaluate how much the severity of histodegeneration is related to the development of neovascularization and to the level of pleiotrophin in the herniated lumbar discs.MethodsSurgically excised lumbar disc specimens were obtained from 29 patients with noncontained (i.e., extruding through the posterior longitudinal ligament) and 21 patients with contained disc herniations. The histodegeneration scores and levels of neovascularization were estimated according to semiquantitative analysis in lumbar disc and endplate samples. Immunohistochemical staining were performed to identify the newly formed blood vessels and to detect the presence of pleiotrophin in the specimens.ResultsHigher levels of disc and endplate neovascularity were registered in noncontained herniations. The level of neovascularization was significantly related to the score of histodegeneration in the herniated disc tissues but not in the endplate specimens. Both contained and noncontained herniations had the highest values of histodegeneration in conjunction with the highest level of neovascularization but the relations between neovascularity and degenerative changes remained to be significant only in the group of noncontained herniations. Registration or frequency of pleiotrophin positive cells did not correlate significantly with histodegeneration or level of neovascularization in the disc samples.ConclusionSevere histodegeneration of the lumbar disc herniations is associated with enhanced neovascularization and potentially also spontaneous regression of the herniated tissue.

Impact of CD133 positive stem cell proportion on survival in patients with glioblastoma multiforme

Abstract Background. The aim of the study was to assess the impact of CD133-positive (CD133+) cancer stem cell proportions on treatment results of glioblastoma multiforme (GBM) patients. Patients and methods. Patients with GBM (n = 42) received postoperative radiotherapy (± chemotherapy). Surgically excised GBM tissue sections were immunohistochemically examined for CD133 expression. The proportions of CD133+ GBM cells were determined (%). The proportion of CD133+ GBM stem cells was established by 2 independent researchers whose results were in good accordance (R = 0.8, p < 0.01). Additionally, CD133 expression levels were correlated with patients overall survival. Results. The proportion of CD133+ cells varied between patients, being from 0.5% to 82%. Mean and median proportions of CD133+ cells of the entire study group were 33% ± 24% (mean ± SD) and 28%, respectively. Clinical data do not support the association between higher proportion of stem cells and the aggressiveness of GBM. Median survival time of the study group was 10.0 months (95% CI 9.0-11.0). The survival time clearly depended on the proportion of CD133+ cells (log rank test, p = 0.02). Median survival times for patients with low (< median) and high (≥ median) proportion of CD133+ cells were 9.0 months (95% CI 7.6-10.5) and 12.0 months (95% CI 9.3-14.7), respectively. In multivariate analysis, the proportion of CD133+ cells emerged as a significant independent predictor for longer overall survival (HR 2.0, 95% CI 1.0-3.8, p = 0.04). Conclusions. In patients with higher stem cell proportion, significantly longer survival times after postoperative radiotherapy were achieved. Underlying reasons and possible higher sensitivity of GBM stem cells to fractionated radiotherapy should be clarified in further studies.

Mechanisms of thyroid hormone control over sensitivity and maximal contractile responsiveness to β-adrenergic agonists in atria

This paper discusses the mechanisms of two basic effects of thyroid hormones on atrial responses to β-adrenergic agonists, i.e. increased inotropic sensitivity and decreased maximal contractile responsiveness. The increased sensitivity of atria to β; adrenergic agonists under thyroid hormones appears to be related to increases in β-adrenoceptor density and Gs/Gi. protein ratio, leading to activation of Gs-mediated pathway, but suppression of Gi.-mediated pathway of adenylate cyclase regulation. Therefore, the i/c concentrations of cAMP and corresponding inotropic responses achieve their maximums at lower doses of β-adrenergic agonist. Thyroid hormones also decrease the expression of phospholamban, but increase the expression of sarcoplasmic reticulum Ca2+-pump. As a result, the basal activity of sarcoplasmic reticulum Ca2+-pump increases, but its β-adrenergic activation through phosphorylation of phospholamban decreases. It is suggested that these changes are causal for decreased maximal inotropic and lusitropic responses of atria to β-adrenergic agonists.(Mol Cell Biochem 184: 419–426, 1998)

Decreased expression of phospholamban is not associated with lower β-adrenergic activation in rat atria

The aim of the study was to find out whether low phospholamban level in atria as compared with ventricles is associated with differences in sarcoplasmic reticular Ca2+-uptake and contractile performance. Relationship between phospholamban and β-adrenergic stimulation in rat left atria and papillary muscles were examined by means of contractile measurements, sarcoplasmic reticular oxalate-supported Ca2+-uptake, and Western blotting of phosphorylated phospholamban. Phosphoprotein determination after β-adrenergic stimulation demonstrated that the levels of Ser16 and Thr17 phosphorylated phospholamban in atria remained at about one-third of that in ventricles. However, comparison of sarcoplasmic reticular Ca2+-uptake in control and isoproterenol perfused preparations demonstrated that the effect of β-adrenergic stimulation on sarcoplasmic reticular Ca2+-uptake was stronger in atrial preparations. Moreover, atria responded to isoproterenol with much larger increases in developed tension, contractility and relaxation rates than papillary muscles. Thus, despite lower level of phospholamban, the β-adrenergic activation of sarcoplasmic reticular Ca2+-uptake and contractile indices are higher in atria.

VEGFR-2 Expression in Glioblastoma Multiforme Depends on Inflammatory Tumor Microenvironment

Glioblastoma multiforme (GBM) is one of the most angiogenic tumors. However, antiangiogenic therapy has not shown significant clinical efficacy. The aim of our study was to evaluate the impact of inflammatory tumor microenvironment on the expression of vascular endothelial growth factor receptor 2 (VEGFR-2). Surgically excised primary GBM tissues were histologically examined for overall extent of inflammation (score 1–3). After immunohistochemistry, the tissue expression of ICAM-1 (optical density), the number of VEGFR-2 positive (VEGFR-2+) blood vessels (per microscopic field), and the endothelial staining intensity of VEGFR-2 (score 0–3) were determined. In GBM, the extent of inflammation was 1.9 ± 0.7 (group mean ± SD). Mean optical density of inflammatory mediator ICAM-1 was 57.0 ± 27.1 (pixel values). The number of VEGFR-2+ blood vessels and endothelial VEGFR-2 staining intensity were 6.2 ± 2.4 and 1.2 ± 0.8, respectively. A positive association was found between endothelial VEGFR-2 staining intensity and the extent of inflammation (p = 0.005). Moreover, VEGFR-2 staining intensity correlated with the expression level of ICAM-1 (p = 0.026). The expression of VEGFR-2, one of the main targets of antiangiogenic therapy, depends on GBM microenvironment. Higher endothelial VEGFR-2 levels were seen in the presence of more pronounced inflammation. Target dependence on inflammatory tumor microenvironment has to be taken into consideration when treatment approaches that block VEGFR-2 signaling are designed.

Bi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia

Infertility affects around 7% of men worldwide. Idiopathic non-obstructive azoospermia (NOA) is defined as the absence of spermatozoa in the ejaculate due to failed spermatogenesis. There is a high probability that NOA is caused by rare genetic defects. In this study, whole-exome sequencing (WES) was applied to two Estonian brothers diagnosed with NOA and Sertoli cell-only syndrome (SCOS). Compound heterozygous loss-of-function (LoF) variants in FANCM (Fanconi anemia complementation group M) were detected as the most likely cause for their condition. A rare maternally inherited frameshift variant p.Gln498Thrfs∗7 (rs761250416) and a previously undescribed splicing variant (c.4387-10A>G) derived from the father introduce a premature STOP codon leading to a truncated protein. FANCM exhibits enhanced testicular expression. In control subjects, immunohistochemical staining localized FANCM to the Sertoli and spermatogenic cells of seminiferous tubules with increasing intensity through germ cell development. This is consistent with its role in maintaining genomic stability in meiosis and mitosis. In the individual with SCOS carrying bi-allelic FANCM LoF variants, none or only faint expression was detected in the Sertoli cells. As further evidence, we detected two additional NOA-affected case subjects with independent FANCM homozygous nonsense variants, one from Estonia (p.Gln1701∗; rs147021911) and another from Portugal (p.Arg1931∗; rs144567652). The study convincingly demonstrates that bi-allelic recessive LoF variants in FANCM cause azoospermia. FANCM pathogenic variants have also been linked with doubled risk of familial breast and ovarian cancer, providing an example mechanism for the association between infertility and cancer risk, supported by published data on Fancm mutant mouse models.

419PASSOCIATION BETWEEN DNA REPAIR ENZYME AND SOMATOSTATIN RECEPTOR IN GLIOBLASTOMA MULTIFORME

ABSTRACT Aim: Glioblastoma multiforme (GBM) by its nature is radio-resistant because with postoperative radiotherapy only short-term stabilization of the disease can be achieved. Prior studies suggest that a higher level of DNA repair enzyme DNA-PK in GBM cells is one cause of the resistance. In current study we tried to identify factors that are associated with higher levels of DNA-PK in GBM. Somatostatin has been shown to be involved in many anti-tumor biological processes, such as inhibition of cell proliferation and the promotion of apoptosis. Therefore, we studied the expression of somatostatin receptor 1 (SSTR1) in GBM tissue and surrounding microenvironment. Methods: Surgically excised GBM tissues (n = 42) were immunohistochemically stained according to standard procedure. Staining intensity of DNA-PK was determined using an arbitrary score (0-3). For SSTR1, the proportion (%) of SSTR-positive (SSTR1+) tumor cells, the number of SSTR1+ blood vessels (per microscopic field) and endothelial SSTR1 staining intensity (score 0-3) were assessed. All individual parameters were characterized by the average value of 10 microscope fields. DNA-PK expression was correlated to SSTR1. Results: All markers were evaluated by two independent investigators whose results were in good accordance (R = 0.8, p Conclusions: The level of DNA repair enzyme DNA-PK in GMB tissue depends on SSTR1 expression in tumor and surrounding microenvironment. The potential of somatostatin in modulating DNA-PK levels and thereby cytotoxic treatment sensitivity has to be clarified in further studies. This work was supported by grant IUT2-4 and by Novartis Pharma unrestricted grant. Disclosure: All authors have declared no conflicts of interest.