Fluorescent microangiography is a novel and widely applicable technique for delineating the renal microvasculature

Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 μm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections.     

Increased potency of vanadium using organic ligands

Thein vivo glucose lowering effect of orally administered inorganic vanadium compounds in diabetes was first reported in our laboratory in 1985. While both vanadate and vanadyl forms of vanadium are orally active, they are still not well absorbed. We have synthesized several organic vanadium compounds and one compound, bis(maltolato)oxovanadium(IV) or BMOV, has been extensively investigated. BMOV proved effective in lowering plasma glucose and lipids in STZ-diabetic rats when administered in drinking water over a 25 week period. The maintenance dose (0.18 mmol/kg/day) was approximately 50% of that required for vanadyl sulfate (VS). Secondary complications of diabetes were prevented by BMOV and no marked toxicity was noted. Oral gavage of STZ-diabetic rats with BMOV also reduced blood glucose levels. The ED₅₀ for BMOV was 0.5 mmol/kg, while for VS the estimated ED₅₀ was 0.9 mmol/kg. BMOV was also effective by the intraperitoneal route in STZ-diabetic rats. The ED₅₀ was 0.08 mmol/kg compared to 0.22 mmol/kg for VS. Some animals treated p.o. or i.p. remained euglycemic for up to 14 weeks. An i.v. infusion of BMOV of 0.05 mmol/kg over a 30 min period reduced plasma glucose levels by 50% while VS was not effective.     

Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion

Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts and lead to heart disease. At present, the source of these cardiac ROS or the subcellular site of cardiac GSH loss [i.e., cytosolic (cGSH) or mitochondrial (mGSH) GSH] has not been completely elucidated. With the use of rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced diabetic rat heart, the mitochondrial origin of ROS was established. Furthermore, mitochondrial damage, as evidenced by loss of membrane potential, increases in oxidative stress, and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and -3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, L-buthionine-sulfoximine (BSO; 10 mmol/kg ip), which blocks GSH synthesis, or diethyl maleate (DEM; 4 mmol/kg ip), which inactivates preformed GSH, was administered in diabetic rats for 4 days after STZ administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were coadministered in diabetic rats. In this setting, mGSH was undetectable and cardiac apoptosis was further aggravated compared with the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification of mGSH in diabetic rat hearts and prevented apoptosis. Our data suggest for the first time that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.     

Effects of chronic administration of clenbuterol on function and metabolism of adult rat cardiac muscle

Clenbuterol (Clen), a beta(2)-agonist, is known to produce skeletal and myocardial hypertrophy. This compound has recently been used in combination with left ventricular assist devices for the treatment of end-stage heart failure to reverse or prevent the adverse effects of unloading-induced myocardial atrophy. However, the mechanisms of action of Clen on myocardial cells have not been fully elucidated. In an attempt to clarify this issue, we examined the effects of chronic administration of Clen on Ca(2+) handling and substrate preference in cardiac muscle. Rats were treated with either 2 mg x kg(-1) x day(-1) Clen or saline (Sal) for 4 wk with the use of osmotic minipumps. Ventricular myocytes were enzymatically dissociated. Cells were field stimulated at 0.5, 1, and 2 Hz, and cytoplasmic Ca(2+) transients were monitored with the use of the fluorescent indicator indo-1 acetoxymethyl ester. Two-dimensional surface area and action potentials in current clamp were also measured. We found that in the Clen group there was significant hypertrophy at the organ and cellular levels compared with Sal. In Clen myocytes, the amplitude of the indo-1 ratio transients was significantly increased. Sarcoplasmic reticulum Ca(2+) content, estimated by rapid application of 20 mM caffeine, was significantly increased in the Clen group. The action potential was prolonged in the Clen group compared with Sal. Carbohydrate contribution to the tricarboxylic cycle (Krebs cycle) flux was increased several times in the Clen group. This increase was associated with decreased expression of peroxisome proliferator-activated receptor-alpha. This study shows that chronic administration of Clen induces cellular hypertrophy and increases oxidative carbohydrate utilization together with an increase in sarcoplasmic reticulum Ca(2+) content, which results in increased amplitude of the Ca(2+) transients. These effects could be important when Clen is used in conjunction with left ventricular assist devices treatment.     

MBEToolbox: a Matlab toolbox for sequence data analysis in molecular biology and evolution

Background  

MATLAB is a high-performance language for technical computing, integrating computation, visualization, and programming in an easy-to-use environment. It has been widely used in many areas, such as mathematics and computation, algorithm development, data acquisition, modeling, simulation, and scientific and engineering graphics. However, few functions are freely available in MATLAB to perform the sequence data analyses specifically required for molecular biology and evolution.