Mechanisms that may be involved in calcium tolerance of the diabetic heart

In diabetes the hearts exhibit impaired membrane functions, but also increased tolerance to Ca²⁺ (iCaT) However, neither the true meaning nor the molecular mechanisms of these changes are fully understood. The present study is devoted to elucidation of molecular alterations, particularly those induced by non-enzymatic glycation of proteins, that may be responsible for iCaT of the rat hearts in the stage of fully developed, but still compensated diabetic cardiomyopathy (DH). Insulin-dependent diabetes (DIA) was induced by a single i.v. dose of streptozotocin (45 mg.kg^-1). Beginning with the subsequent day, animals obtained 6 U insulin daily. Glucose, triglycerides, cholesterol and glycohemoglobin were investigated in blood. ATPase activities, the kinetics of activation of (Na,K)-ATPase by Na⁺ and K⁺, further the fluorescence anisotropy of diphenyl-hexatriene as well as the order parameters of membranes in isolated heart sarcolemma (SL) were also investigated. In addition, the degree of glycation and glycation-related potency for radical generation in SL proteins were determined by investigating their fructosamine content. In order to study calcium tolerance of DH in a 'transparent' model, hearts were subjected to calcium paradox (Ca-Pa, 3 min of Ca²⁺ depletion; 10 min of Ca²⁺ repletion). In this model of Ca²⁺-overload, Ca²⁺ ions enter the cardiac cells in a way that is not mediated by receptors. Results revealed that more than 83% of the isolated perfused DH recovered, while the non-DIA control hearts all failed after Ca-Pa. DH exhibited well preserved SL ATPase activities and kinetics of (Na,K)-ATPase activation by Na⁺, even after the Ca-Pa. This was considered as a reason for their iCaT. Pretreatment and administration of resorcylidene aminoguanidine (RAG 4 or 8 mg.kg^-1) during the disease prevented partially the pathobiochemical effects of DIA-induced glycation of SL proteins. DIAinduced perturbations in anisotropy and order parameters of SL were completely prevented by administration of RAG (4 mg.kg^-1). Although, the latter treatment exerted little influence on the (Na,K)-ATPase activity, it decreased the calcium tolerance of the DH. Results are supporting our hypothesis that the glycation-induced enhancement in free radical formation and protein crosslinking in SL may participate in adaptive mechanisms that may be also considered as 'positive' and are responsible for iCaT of the DH.     

Homodimerization of the Wnt Receptor DERAILED Recruits the Src Family Kinase SRC64B

Ryk pseudokinase receptors act as important transducers of Wnt signals, particularly in the nervous system. Little is known, however, of their interactions at the cell surface. Here, we show that a Drosophila Ryk family member, DERAILED (DRL), forms cell surface homodimers and can also heterodimerize with the two other fly Ryks, DERAILED-2 and DOUGHNUT ON 2. DERAILED homodimerization levels increase significantly in the presence of its ligand, WNT5. In addition, DERAILED displays ligand-independent dimerization mediated by a motif in its transmembrane domain. Increased dimerization of DRL upon WNT5 binding or upon the replacement of DERAILED''s extracellular domain with the immunoglobulin Fc domain results in an increased recruitment of the Src family kinase SRC64B, a previously identified downstream pathway effector. Formation of the SRC64B/DERAILED complex requires SRC64B''s SH2 domain and DERAILED''s PDZ-binding motif. Mutations in DERAILED''s inactive tyrosine kinase-homologous domain also disrupt the formation of DERAILED/SRC64B complexes, indicating that its conformation is likely important in facilitating its interaction with SRC64B. Finally, we show that DERAILED''s function during embryonic axon guidance requires its Wnt-binding domain, a putative juxtamembrane extracellular tetrabasic cleavage site, and the PDZ-binding domain, indicating that DERAILED''s activation involves a complex set of events including both dimerization and proteolytic processing.     

Biological activities of (−)-epicatechin and (−)-epicatechin-containing foods: Focus on cardiovascular and neuropsychological health

Recent studies have suggested that certain (?)-epicatechin-containing foods have a blood pressure-lowering capacity. The mechanisms underlying (?)-epicatechin action may help prevent oxidative damage and endothelial dysfunction, which have both been associated with hypertension and certain brain disorders. Moreover, (?)-epicatechin has been shown to modify metabolic profile, blood's rheological properties, and to cross the blood-brain barrier. Thus, (?)-epicatechin causes multiple actions that may provide unique synergy beneficial for cardiovascular and neuropsychological health. This review summarises the current knowledge on the biological actions of (?)-epicatechin, related to cardiovascular and brain functions, which may play a remarkable role in human health and longevity.     

Neopterin in renal cell carcinoma: inhalational administration of interleukin-2 is not accompanied by a rise of urinary neopterin.

Inhalational administration of interleukin-2 (IL-2) is effective in controlling renal cell carcinoma (RCC) lung metastases with minimal toxicity. Neopterin is an indicator of systemic immune activation in metastatic cancer and is increased after systemic IL-2 administration. Urinary neopterin was investigated in 13 patients with metastatic RCC and 18 controls. In seven patients, urinary neopterin was followed before and after treatment with inhalational IL-2. Neopterin was measured by high-performance liquid chromatography and creatinine was determined by Jaffé reaction. Urinary neopterin was significantly increased in patients with metastatic RCC compared to controls (257 +/- 263 micromol/mol creatinine vs. 110 +/- 41 micromol/mol creatinine; Mann-Whitney U-test, p < 0.05). Median survival was significantly longer in patients with urinary neopterin <173 micromol/mol creatinine compared to patients with neopterin > or = 173 micromol/mol creatinine (698 vs. 245 days; log-rank test, p < 0.05). No significant increase was observed after inhalational IL-2 therapy (147 +/- 101 vs. 153 +/- 54 micromol/mol creatinine). We conclude that urinary neopterin is increased in patients with metastatic RCC, and higher neopterin concentrations are indicative of poor prognosis. The absence of an increase in urinary neopterin after inhalational IL-2 therapy is in accord with the lack of significant systemic toxicity.     

Survival of Patients with Primary Brain Tumors: Comparison of Two Statistical Approaches

Purpose

We reviewed the survival time for patients with primary brain tumors undergoing treatment with stereotactic radiation methods at the Masaryk Memorial Cancer Institute Brno. We also identified risk factors and characteristics, and described their influence on survival time.

Methods

In summarizing survival data, there are two functions of principal interest, namely, the survival function and the hazard function. In practice, both of them can depend on some characteristics. We focused on nonparametric methods, propose a method based on kernel smoothing, and compared our estimates with the results of the Cox regression model. The hazard function is conditional to age and gross tumor volume and visualized as a color-coded surface. A multivariate Cox model was also designed.

Results

There were 88 patients with primary brain cancer, treated with stereotactic radiation. The median survival of our patient cohort was 47.8 months. The estimate of the hazard function has two peaks (about 10 months and about 40 months). The survival time of patients was significantly different for various diagnoses (p≪0.001), KI (p = 0.047) and stereotactic methods (p = 0.033). Patients with a greater GTV had higher risk of death. The suitable threshold for GTV is 20 cm³. Younger patients with a survival time of about 50 months had a higher risk of death. In the multivariate Cox regression model, the selected variables were age, GTV, sex, diagnosis, KI, location, and some of their interactions.

Conclusion

Kernel methods give us the possibility to evaluate continuous risk variables and based on the results offer risk-prone patients a different treatment, and can be useful for verifying assumptions of the Cox model or for finding thresholds of continuous variables.     

Investigation of Paradiplozoon homoion (Monogenea, Diplozoidae) life cycle under experimental conditions

Diplozoids (Diplozoidae, Monogenea) are fish ectoparasites with a direct life cycle without intermediate hosts. Their free swimming larva, the oncomiracidium, hatches from eggs, invades a fish host and metamorphoses into a post-oncomiracidial larval stage, the diporpa. Later, two diporpae fuse and live as a pair in cross-copulation during their adult life. An experimental study was designed to investigate the life cycle of Paradiplozoon homoion (Monogenea, Diplozoidae) parasitizing their common fish hosts, gudgeon (Gobio gobio). A total of 35 gudgeon parasitized by diplozoids were collected from their natural environment of the Vlára River, Czech Republic, and kept together in tanks with 41 non-parasitized gudgeons reared in a laboratory environment. In total, 100 adult specimens of P. homoion were collected from the Vlára River gudgeon and a new parasite generation was expected to be observed on fish reared in the laboratory environment. Eight days after the first diplozoid eggs appeared on fish gills, the presence of diporpae with one or two pairs of clamps was noted. The appearance of the first juveniles was recorded at the same time as diporpae. Development of P. homoion from egg to sexually mature adult stage took 33 days at a constant temperature of 20 degrees C. The development of eggs in adults of the second generation was observed 2 days after the first observation of these adults. The behavior of oncomiracidia was also studied and this free swimming stage of diplozoids survived for 22 h in the absence of a host. When host fish were experimentally infected by oncomiracidia, diporpae were found attached to the fish gill apparatus within 2 h of infection.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.     

Inhibitory Effects of Some Spice and Herb Extracts Against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii

Seventeen spice and medicinal plant extracts (methanol and chloroform) were assayed for their antimicrobial activity against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii. In general, all of the tested extracts were able, to a different extent, to inhibit the growth of the selected Arcobacter species. Cinnamon, bearberry, chamomile, sage and rosemary extracts showed strong antimicrobial activity toward arcobacter strains tested. Overall, the methanol extracts showed better activity than the chloroform extracts (P < 0.05); however, enhanced antibacterial activity of chloroform extracts of cinnamon and rosemary has been observed in comparison with their methanol counterparts. The inhibitory dose of the most active extracts (the diameter of zone of inhibition ≥ 20 mm) was determined using the disc-diffusion method as well.     

New member of the protein disulfide isomerase (PDI) family identified in Amblyomma variegatum tick

Ticks belonging to arthropoda are blood feeding, geographically widespread ectoparasites of mammals, reptiles and birds. Their saliva contains active substances that protect them from host immune attack and allow for transmission of various pathogens during the feeding process. Characterization of tick saliva components can therefore contribute to the development of effective methods for the control of tick-borne diseases.

Here we describe the identification and basic characterization of a gene encoding a 55 kDa protein found in the salivary glands (SG) of Amblyomma variegatum tick. Based on the primary structure and homology to the family of protein disulfide isomerases (PDI; EC 5.3.4.1) the gene was named AvPDI. The 1461 nt long AvPDI open reading frame codes for a 487 amino acid protein. In vitro expressed AvPDI was exclusively localized in the endoplasmic reticulum. RT-PCR and Western blot analysis revealed that AvPDI expression is not restricted to the SG of the tick. More detailed analysis on tissue slides from SG detected an AvPDI specific signal in granular cells of the acini type II and III. Finally, reductase activity of AvPDI was confirmed in an insulin assay. The structural and functional characteristics suggest that AvPDI is another member of the PDI protein family and represents the first more closely characterized PDI in the ticks.