Trandolapril and endothelin antagonist LU-135252 in the treatment of the fructose-induced hypertensive,hyperinsulinemic, hypertriglyceridemic rat

BACKGROUND: In view of the demonstrated interaction between endothelin and the renin-angiotensin system, the antihypertensive effect of combined therapy with an endothelin antagonist LU-135252 and the angiotensin converting enzyme inhibitor trandolapril, was studied in fructose-induced hypertensive, hyperinsulinemic, hypertriglyceridemic male Sprague-Dawley rats. METHODS: Forty animals were fed a fructose-enriched diet (Tekled, Harlan) for 5 weeks, as follows: group A, fructose only; group B, trandolapril 0.1 mg/kg/day added during the last 2 weeks; group C, LU-135252 100 mg/kg/day added during the last 2 weeks; group D, both trandolapril and LU-135252 added the last 2 weeks. Systolic blood pressure (BP) was measured weekly in conscious rats by the indirect tail-cuff method. Blood samples from a retro-orbital sinus puncture were taken at the beginning of the experiment and after 3 and 5 weeks and examined for insulin and triglyceride concentrations. RESULTS: Systolic BP decreased in group B (trandolapril) from 148.8 +/- 9.8 at 3 weeks to 138.3 +/- 8.7 mm Hg after 5 weeks; in group C (endothelin antagonist) from 155.1 +/- 5.5 to 142.5 +/- 10.6 mm Hg; and in group D (combination) from 154.6 +/- 10.9 to 121.2 +/- 8.9 mm Hg. Triglyceride levels decreased only in the combined trandolapril/endothelin antagonist group from 167.6 +/- 55.3 in the third week to 134.9 +/- 53.7 mg/dL after 5 weeks. Insulin levels decreased only on combination therapy from 7.4 +/- 3.6 to 5.3 +/- 3.8 ng/mL during the same period. The BP decrease was additive compared with the respective individual substances. CONCLUSIONS: The trandolapril/endothelin antagonist combination appears to offer a rational antihypertensive combination that is superior to that of either drug alone. This finding applies to the specific rat model studied in which BP, insulin, and triglycerides were increased by fructose diet.     

MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi

Lyme borreliosis, caused by the tick-borne bacterium Borrelia burgdorferi, has become the most common vector-borne disease in North America over the last three decades. To understand the dynamics of the epizootic spread and to predict the evolutionary trajectories of B. burgdorferi, accurate information on the population structure and the evolutionary relationships of the pathogen is crucial. We, therefore, developed a multilocus sequence typing (MLST) scheme for B. burgdorferi based on eight chromosomal housekeeping genes. We validated the MLST scheme on B. burgdorferi specimens from North America and Europe, comprising both cultured isolates and infected ticks. These data were compared with sequences for the commonly used genetic markers rrs-rrlA intergenic spacer (IGS) and the gene encoding the outer surface protein C (ospC). The study demonstrates that the concatenated sequences of the housekeeping genes of B. burgdorferi provide highly resolved phylogenetic signals and that the housekeeping genes evolve differently compared with the IGS locus and ospC. Using sequence data, the study reveals that North American and European populations of B. burgdorferi correspond to genetically distinct populations. Importantly, the MLST data suggest that B. burgdorferi originated in Europe rather than in North America as proposed previously.     

An Analysis of How to Measure Glucose during Glucose Clamps: Are Glucose Meters Ready for Research?

This article provides a perspective on the challenges of appropriate glucose measurement in the context of glucose clamp experiments. In a first step, the core outcome parameters of a clamp experiment, the blood glucose target level, and the glucose infusion rate will be identified. The relation of these core parameters to glucose measurement are discussed. From there, the core quality parameters of glucose measurement within a clinical research setting are identified and assessed in light of their practical implications, with a specific consideration of the work presented by Cohen et al. in this issue of the Journal of Diabetes Science and Technology.     

Automatic tracking of rolling leukocytes in vivo.

The analysis of instantaneous and average rolling leukocyte velocity is crucial to the study of inflammatory disease. In order to record features associated with leukocyte rolling, the leukocyte position must be tracked, typically by manual observation. Automated tracking of leukocytes is possible for in vitro studies, but not for recordings resulting from intravital experiments. Therefore, we have designed and implemented an image processing system for automated tracking of rolling leukocytes in vivo. The novel image processing techniques used in the tracking system successfully address the four major problems associated with tracking cells in vivo: background movement, severe image noise and clutter, cell deformation and contrast change, and occlusion of the target cell by other structures. We have tested the system in two experimental protocols in which leukocyte rolling is observed in venules of the mouse cremaster muscle with and without TNF-alpha treatment. The automated tracking system was validated by comparing automatically generated displacement and velocity data with data from the same recordings collected manually. The root mean squared error between the computed displacements and the manually measured displacements was less than 12% of the average displacement in TNF-alpha-treated venules. The average velocity error was also less than 12%. For untreated venules, the computed and measured displacements and velocities had an RMSE of less than 8%. The automated tracking system allows one, for the first time, to reliably track rolling leukocytes in vivo, thus eliminating possible investigator bias and increasing throughput.     

Increased amount of a mitochondria-associated 21 x 103 dalton protein in human gastrointestinal tumors

Summary Differences in the structure and number of mitochondria in tumor cells were found. Using isoelectrofocussing two-dimensional polyacrylamide gel electrophoresis which allows detection of alterations in the protein pattern of tumor mitochondria, we studied both quantitative and qualitative changes in the mitochondrial protein pattern of human gastrointestinal tumors and corresponding normal matrix tissues. One low molecular protein spot was found to be quantitatively changed in the tumors. The approximate molecular weight was 21 x 103 daltons and the pl value 5.7.Dedicated to Dr. Hellmuth Sitte on the occasion of his 60th birthday     

Oral cyclosporin-A is effective treatment for untreated and also for previously immunosuppressed patients with severe bone marrow failure

16 patients with transfusion-dependent, life-threatening bone marrow failure (14 with severe aplastic anaemia, 1 with systemic lupus erythematosus and 1 with pure red cell aplasia) were treated with cyclosporin-A (Cy-A) after either lack of response to conventional immunosuppression with antithymocyte-globulin/high-dose methylprednisolone for 95 to 1190 days (median 186.5) (group I, 8 patients) or as a primary treatment due to ineligibility for conventional immunosuppression (group II, 8 pat.). Cyclosporin-A was given orally to maintain trough levels of 200 to 300 ng/ml (RIA). In group I, 6 out of 8 patients responded 30 to 480 d (median 53) and are currently alive 627 to 1482 d (median 731) after initiation of Cy-A, respectively. In 3 of the responders Cy-A has been withdrawn, without relapse. In group II, 5 of 8 patients responded 26 to 170 d (median 63) and are currently alive 142 to 697 (median 420) d following initiation of Cy-A, respectively. These data indicate a place for cyclosporin-A in the management of patients with life-threatening bone marrow failure in whom a) immunosuppressive therapy with antithymocyte-globulin and high-dose methylprednisolone had failed and b) who are not candidates for vigorous immunosuppression or bone marrow transplantation, for medical or other reasons.     

Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms

The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases’ homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.Since its discovery, c-Src (1) has been subject to intensive research into its cellular functions and regulation. Whereas c-Src is the best-studied protooncogene, less is known about the other, closely related Src family kinase (SFK) members. Their high degree of similarity in structure and regulation suggests that SFKs can partially compensate for each other in vivo. Indeed, knockout studies have shown that only mice deficient in all three genes (src, yes, and fyn) show embryonic lethality (2). Early studies demonstrated that disruption of Src or Fyn genes individually resulted only in subtle changes in function of a few cell types (e.g., osteoclasts for src^−/−, and T cells for fyn^−/−) (3, 4). Roche et al. provided strong evidence that Src, Yes, and Fyn substitute for each other during cell cycle progression (5). These studies suggested that there is a high degree of functional redundancy among Src family kinases.Nonetheless, emerging evidence indicates that Src and Fyn regulate distinct processes in the same cell. Down-regulation of Fyn expression enhances VEGF-stimulated migration of endothelial cells, whereas down-regulation of Src does not (6). Differences in the transforming capacity of SFKs are thought to depend on their affinity for cholesterol-enriched membrane microdomains, which is determined in part by their N-terminal lipid modifications (7, 8). Src has higher tumorigenic potential than Fyn in prostate epithelium, and this is differently affected by alterations in N-terminal palmitoylation (9). Previous studies have shown that Src localizes to perinuclear endosomal compartments and translocates to the plasma membrane upon activation (10–12), whereas Fyn localizes to the plasma membrane regardless of its activity (13, 14). Although these studies suggest that localization is important in differentiating the actions of the two kinases, they do not identify specific roles associated with particular subcellular locations.Various techniques have been applied to elucidate the differences in signaling specificity of SFKs. Kinase–substrate interactions have been examined using purified substrates (15). Mutated kinases with selectivity for radiolabeled ATP analogs have identified directly phosphorylated substrates of Src (16). These methods were restricted to cell lysates or purified proteins, and so were unable to address the role of cellular localization in substrate specificity.To dissect the unique role of different SFK isoforms (2–4, 17, 18) in living cells, we engineered regulatable analogs of Fyn, Yes, and LynA kinases using our rapamycin-regulated activation (RapR) strategy, which has been developed using Src as a prototype (19, 20). Insertable FKBP12 (iFKBP, a truncated form of FKBP) was inserted into the catalytic domain of each SFK, which abolished their kinase activity. Activity was rescued by treating cells with rapamycin in the presence of the FKBP12-rapamycin binding domain (FRB) (Fig. 1A). Molecular dynamics studies have indicated that heterodimerization of the inserted iFKBP with FRB likely reduces the conformational mobility of the kinase G loop, restoring ATP binding (3, 21).Open in a separate windowFig. 1.Design of RapR kinases. (A) Schematic representation of the approach used to regulate catalytic activity of SFKs. The insertion of iFKBP at a highly conserved site in the catalytic domain of each kinase resulted in loss of kinase activity. Catalytic activity was restored by rapamycin, which induced binding of iFKBP and coexpressed FRB. (B) Sequence alignment of SFKs shows that there is a well-defined loop where iFKBP is inserted (blue). It is linked to the G loop (red) through a β-sheet in each SFK.These analogs enabled activation of each isoform specifically, within minutes, resulting in clear phenotypic differences. Unlike genetic modifications of cell populations, there was little time for the cell to compensate for kinase activation before observation. The induced cell behaviors occurred in a succession of stages, associated with changes in the subcellular distribution of each kinase. We focused on Src and Fyn, developing quantitative tools to carefully characterize the kinetics of induced behaviors and associated localization dynamics. Our results indicated that Src’s unique ability to induce polarized movement shortly after kinase activation results from its localization in a perinuclear compartment, where it phosphorylates substrates that traffic on microtubules to the cell perimeter. Both the localization dynamics and phenotype differences between Src and Fyn were dependent on N-terminal lipid modifications, and not on SH2 and SH3 domain interactions.     

“ToxRTool”, a new tool to assess the reliability of toxicological data

Evaluation of the reliability of toxicological data is of key importance for regulatory decision-making. In particular, the new EU Regulations concerning the registration, evaluation, authorisation and restriction of chemicals (REACH) and classification, labelling and packaging (CLP) according to the new globally harmonised system (GHS) rely on the integration of all available toxicological information. The so-called Klimisch categories, although well established and widely used, lack detailed criteria for assigning data quality to categories. A software-based tool (ToxRTool) was developed within the context of a project funded by the European Commission to provide comprehensive criteria and guidance for reliability evaluations of toxicological data. It is applicable to various types of experimental data, endpoints and studies (study reports, peer-reviewed publications) and leads to the assignment to Klimisch categories 1, 2 or 3. The tool aims to increase transparency and to harmonise approaches of reliability assessment. The tool consists of two parts, one to evaluate in vivo and one to evaluate in vitro data. The prototypes of the tool were tested in two independent inter-rater experiments. This approach allowed the analysis of the performance of the tool in practice and the identification and minimisation of sources of heterogeneity in evaluation results. The final version, ToxRTool, is publicly available for testing and use.