Rapid and sensitive detection of benzodiazepines and zopiclone in serum using high-performance thin-layer chromatography

We developed a rapid and sensitive method of identifying benzodiazepines and zopiclone in human serum using high-performance thin-layer chromatography (HPTLC). These drugs were developed and separated on plates within 8–11 min and detected by means of UV radiation and colour. Each drug was accurately identified by means of the values of R_F × 100 and the spot colour in three systems. The detection limit of the benzodiazepines in serum was 0.1-0.4 μg/ml, except for cloxazolam and haloxazolam. The sensitivity was increased about ten-fold over the conventional method. These results suggested that the HPTLC system is useful for the initial detection and identification of these drugs in emergencies.     

Endothelial dysfunction and hypercontractility of vascular myocytes are ameliorated by fluvastatin in obese Zucker rats

Static exercise causes activation of the sympathetic nervous system, which results in increased blood pressure (BP) and renal vascular resistance (RVR). The question arises as to whether renal vasoconstriction that occurs during static exercise is due to sympathetic activation and/or related to a pressure-dependent renal autoregulatory mechanism. To address this issue, we monitored renal blood flow velocity (RBV) responses to two different handgrip (HG) exercise paradigms in 7 kidney transplant recipients (RTX) and 11 age-matched healthy control subjects. Transplanted kidneys are functionally denervated. Beat-by-beat analyses of changes in RBV (observed via duplex ultrasound), BP, and heart rate were performed during HG exercise in all subjects. An index of RVR was calculated as BP/RBV. In protocol 1, fatiguing HG exercise (40% of maximum voluntary contraction) led to significant increases in RVR in both groups. However, at the end of exercise, RVR was more than fourfold higher in control subjects than in the RTX group (88 vs. 20% increase over baseline; interaction, P < 0.001). In protocol 2, short bouts of HG exercise (15 s) led to significant increases in RVR at higher workloads (50 and 70% of maximum voluntary contraction) in the control subjects (P < 0.001). RVR did not increase in the RTX group. In conclusion, we observed grossly attenuated renal vasoconstrictor responses to exercise in RTX subjects, in whom transplanted kidneys were considered functionally denervated. Our results suggest that renal vasoconstrictor responses to exercise in conscious humans are mainly dependent on activation of a neural mechanism.     

Cardiovascular proteomic analysis

Here, we report on our proteomic studies in the field of cardiovascular medicine. Our research has been focused on understanding the role of proteins in cardiovascular disease with a particular focus on epigenetic regulation and biomarker discovery, with the objective of better understanding cardiovascular pathophysiology to lead to the development of new and better diagnostic and therapeutic methods. We have used mass spectrometry for over 5 years as a viable method to investigate protein-protein interactions and post-translational modifications in cellular proteins as well as a method to investigate the role of extra-cellular proteins. Use of mass spectrometry not only as a research tool but also as a potential diagnostic tool is a topic of interest. In addition to these functional proteomics studies, structural proteomic studies are also done with expectations to allow for pinpoint drug design and therapeutic intervention. Collectively, our proteomics studies are focused on understanding the functional role and potential therapeutically exploitable property of proteins in cardiovascular disease from both intra-cellular and extra-cellular aspects with both functional as well as structural proteomics approaches to allow for comprehensive analysis.     

IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs

Intravital visualization of thrombopoiesis revealed that formation of proplatelets, which are cytoplasmic protrusions in bone marrow megakaryocytes (MKs), is dominant in the steady state. However, it was unclear whether this is the only path to platelet biogenesis. We have identified an alternative MK rupture, which entails rapid cytoplasmic fragmentation and release of much larger numbers of platelets, primarily into blood vessels, which is morphologically and temporally different than typical FasL-induced apoptosis. Serum levels of the inflammatory cytokine IL-1α were acutely elevated after platelet loss or administration of an inflammatory stimulus to mice, whereas the MK-regulator thrombopoietin (TPO) was not elevated. Moreover, IL-1α administration rapidly induced MK rupture–dependent thrombopoiesis and increased platelet counts. IL-1α–IL-1R1 signaling activated caspase-3, which reduced plasma membrane stability and appeared to inhibit regulated tubulin expression and proplatelet formation, and ultimately led to MK rupture. Collectively, it appears the balance between TPO and IL-1α determines the MK cellular programming for thrombopoiesis in response to acute and chronic platelet needs.     

Administration of ANG II induces iron deposition and upregulation of TGF-beta1 mRNA in the rat liver

We previously found that ANG II infusion into rats causes iron deposition in the kidney and heart, which may have a role in the regulation of profibrotic gene expression and tissue fibrosis. In the present study, we have investigated whether ANG II can also induce iron accumulation in the liver. Prussian blue staining detected frequent iron deposition in the interstitium of the liver of rats treated with pressor dose ANG II for 7 days, whereas iron deposition was absent in the livers of control rats. Immunohistochemical and histological analyses showed that some iron-positive nonparenchymal cells were positive for ferritin and heme oxygenase-1 (HO-1) protein and TGF-beta1 mRNA and were judged to be monocytes/macrophages. It was shown that ANG II infusion caused about a fourfold increase in ferritin and HO-1 protein expression by Western blot analysis and about a twofold increase in TGF-beta1 mRNA expression by Northern blot analysis, which were both suppressed by treating ANG II-infused rats with losartan and deferoxamine. In addition, mild interstitial fibrosis was observed in the liver of rats that had been treated with pressor dose ANG II for 7 days or with nonpressor dose ANG II for 30 days, the latter of which also caused loss of hepatocytes and intrahepatic hemorrhage in the liver. Taken together, our data suggest that ANG II infusion induces aberrant iron homeostasis in the liver, which may have a role in the ANG II-induced upregulation of profibrotic gene expression in the liver.     

Single cell mechanics of rat cardiomyocytes under isometric, unloaded, and physiologically loaded conditions

One of the most salient characteristics of the heart is its ability to adjust work output to external load. To examine whether a single cardiomyocyte preparation retains this property, we measured the contractile function of a single rat cardiomyocyte under a wide range of loading conditions using a force-length measurement system implemented with adaptive control. A pair of carbon fibers was used to clamp the cardiomyocyte, attached to each end under a microscope. One fiber was stiff, serving as a mechanical anchor, while the bending motion of the compliant fiber was monitored for force-length measurement. Furthermore, by controlling the position of the compliant fiber using a piezoelectric translator based on adaptive control, we could change load dynamically during contractions. Under unloaded conditions, maximal shortening velocity was 106 +/- 8.9 microm/s (n = 13 cells), and, under isometric conditions, peak developed force reached 5,720 nN (41.6 +/- 5.6 mN/mm(2); n = 17 cells). When we simulated physiological working conditions consisting of an isometric contraction, followed by shortening and relaxation, the average work output was 828 +/- 123 J/m(3) (n = 20 cells). The top left corners of tension-length loops obtained under all of these conditions approximate a line, analogous to the end-systolic pressure-volume relation of the ventricle. All of the functional characteristics described were analogous to those established by studies using papillary muscle or trabeculae preparations. In conclusion, the present results confirmed the fact that each myocyte forms the functional basis for ventricular function and that single cell mechanics can be a link between subcellular events and ventricular mechanics.