The Signature Program: Bringing the Protocol to the Patient.

Early-phase clinical development in oncology has evolved dramatically with the deciphering of the human genome in 2004. Genomic analysis and the tools identifying genetically disrupted pathways within a patient's tumor have been a driving force for personalized medicine and for the development of highly targeted novel therapies. Tumors are often genetically heterogeneous, with multiple concurrent genetic abnormalities. On the other hand, tumors arising from different tissues may share identical molecular drivers.

Ultrastructural examination of rabbit aortic wall following high-fat diet feeding and selenium supplementation: a transmission electron microscopy study.

Experiments were carried out to examine the changes occurring in the wall of rabbit aortae following high-fat diet (HFD) feeding as well as HFD + selenium supplementation. Male New Zealand White rabbits were divided into three groups-control, HFD-fed and HFD + Se supplementation-and were treated for three months. The study depicted that levels of serum total cholesterol and triglycerides were markedly increased in the HFD-fed group as compared with control animals. However, in the HFD + Se-fed group, these levels were markedly suppressed vis-à-vis animals fed on HFD only. Development of atherogenic and atheromatic plaques has been shown at the light microscopy level in HFD-fed rabbits, whereas these developments were not visible in the HFD + Se-fed rabbits. Transmission electron microscopy findings indicated altered ultrastructure in the endothelial cells of the intimal layer as well as smooth-muscle cells of the medial layer in HFD-fed animals. However, these findings indicated normal ultrastructure in most of the cells, with little ultrastructural alterations from animals supplemented with Se along with HFD feeding. The study on the whole depicted the ability of Se to inhibit the onset of progression of aortic disease and hence has relevance to its therapeutic potential.

Studies of apoptosis and bcl-2 in experimental atherosclerosis in rabbit and influence of selenium supplementation.

Apoptosis is a ubiquitous physiological mechanism of cell death regulating tissue mass and architecture. An attempt was made in the present study to see the occurrence of apoptotic cell death in three different treatment groups of rabbits viz. Control, HFD fed and HFD + Selenium fed. Apoptotic activity as checked by in situ DNA end labelling (TUNEL Assay) revealed excessive staining, mostly concentrated in plaque region both in fibrous as well as atheromatous plaque in HFD fed animals. However, in selenium (Se) supplemented animals, very little TUNEL staining could be seen, and even that confined to endothelial cells only. The control group on the other hand was totally devoid of any staining. Transmission Electron Microscopic (TEM) study also depicted the occurrence of apoptosis in aortic cells of HFD fed animals and very little in Se supplemented animals. Apoptotic activity has been discussed in relation to oxidative stress in HFD fed group. bcl-2, though an antiapoptotic oncoprotein, was found to be expressed more in atherogenic group as compared to control/HFD + Se treatment. On the whole, the study highlighted the occurrence of apoptotic process in atherosclerosis and the role of Se, a potent antioxidant, in inhibition of apoptotic process in HFD fed animals.

Molecular biology of protein kinase C signaling in cardiac myocytes.

The PKC family of serine/threonine kinases have been implicated in a diverse array of cellular responses. Adult cardiac myocytes express multiple PKC isozymes, which participate in the response of muscle cells to extracellular stimuli, modulate contractile properties, and promote cell growth and survival. Recently, the classification of this ubiquitous family of signaling molecules has been expanded from three to four subfamilies. This review will focus on the application of pharmacologic and molecular approaches to explore the biology of cardiac PKC isozymes. The availability of transgenic mice and peptide PKC modulators have been instrumental in identifying target substrates for activated cardiac PKC isozymes, as well as the identification of specific isozymes linked to distinct growth characteristics and cell phenotype. The rapid growth of knowledge in the area of PKC signaling and PKC substrate interactions, may result in the development of therapeutic modalities with the potential to arrest or reverse the progression of cardiovascular diseases.

Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I.

Activation of the protein kinase C (PKC) family is a potential signaling mechanism by which high ambient glucose concentration modulates the phenotype and physiological function of cells. Recently, the cardiac renin angiotensin system (RAS) has been reported to promote PKC translocation in the diabetic heart via the angiotensin (ANG) II type 1 receptor (AT-1R). To evaluate the molecular events coupled with high glucose-induced PKC translocation and to examine the role of endogenously released ANG II in myocyte PKC signaling, primary cultures of adult rat ventricular myocytes were exposed to normal (5 mmol/l) or high (25 mmol/l) glucose for 12-24 h. Western blot analysis indicated that adult rat ventricular myocytes coexpress six PKC isozymes (alpha, beta(1,) beta(2,) delta, epsilon, and zeta). Translocation of five PKC isozymes (beta(1), beta(2), delta, epsilon, and zeta) was detected in response to 25 mmol/l glucose. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate blocked glucose-induced translocation of PKC-beta(2), -delta, and -zeta. Inhibition of tyrosine kinase with genistein blocked glucose-induced translocation of PKC-beta(1) and -delta, whereas chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane N,N,N,'N'-tetraacetic acid blocked translocation of PKC-beta(1) and -beta(2). Enzyme-linked immunosorbent assay performed on culture media from myocytes maintained in 25 mmol/l glucose detected a twofold increase in ANG II. Addition of an AT-1R antagonist (losartan; 100 nmol/l) to myocyte cultures blocked translocation of PKC-beta(1), -beta(2), -delta, and -epsilon. Phosphorylation of troponin (Tn) I was increased in myocytes exposed to 25 mmol/l glucose. Losartan selectively inhibited Tn I serine phosphorylation but did not affect phosphorylation at threonine residues. We concluded that 1) 25 mmol/l glucose triggers the release of ANG II by myocytes, resulting in activation of the ANG II autocrine pathway; 2) differential translocation of myocyte PKC isozymes occurs in response to 25 mmol/l glucose and ANG II; and 3) AT-1R-dependent PKC isozymes (beta(1), beta(2), delta, and epsilon) target Tn I serine residues.

Selenium supplementation protects from high fat diet-induced atherogenesis in rats: role of mitogen stimulated lymphocytes and macrophage NO production.

The present study was designed to demonstrate the involvement of immune response in experimental atherogenesis. The mitogenic stimulation of lymphocytes and NO production by macrophages in experimental atherogenesis were studied. Further, influence of selenium a potent antioxidant was also studied in the disease process. Three different treatment groups of rats undertaken for study were: group 1, control; group II, high fat diet (HFD) fed group and group III, HFD+Se supplemented group. Atherogenic conditions induced have already been explained earlier [Kang BPS et al. Gen Physiol Biophys, 17 (1998) 71]. Significant increase in 3H-thymidine incorporation was obtained in lymphocytes from HFD fed animals in both presence and absence of mitogen (Con-A). However, these values decreased in group III animals, which were supplemented with selenium. Similarly, NO levels with LPS+ and LPS- macrophages also found to be higher in HFD fed group and decreased in group III. These studies reveal the protective role of selenium in HFD-induced atherogenic process.

Molecular and kinetic properties of sperm specific LDH after radiation inactivation.

Radiation inactivation of sperm specific lactate dehydrogenase-C4 (LDH-C4) has been studied and compared with the somatic LDH in aqueous solution. D37 of C isozyme was 470 Gy and that of B isozyme was 520 Gy. Semi-log plots of log N/No versus dose suggested that the inactivation of two LDH isozymes in presence of normal saline follows a single hit kinetics. Target molecular weight calculated by radiation analysis was found as 1.52 x 10(5) gm/mole for LDH-C4 and 1.38 x 10(5) gm/mole for LDH-B4. SDS-PAGE of irradiated enzymes showed a band of 35 kDa but did not indicate the presence of any other extra band, when compared with sham-irradiated enzymes. Chemical kinetics of residual activity following irradiation at D37 showed decrease in Vmax with coenzymes and primary substrates. However, decrease in Km was seen with pyruvate as increasing substrate. Nevertheless, K did not change when NAD+ was the leading substrate for LDH-B4 or LDH-C4. A hyperchromicity in intrinsic fluorescence and a blue shift in lambdamax over sham-irradiated LDH-C4 revealed the exposure of buried tryptophan residues to the surface after radiation inactivation. Results suggest that inspite of presence of variant amino acids, the conformations of two isozymes are stabilized by similar forces which behave in a similar way for radiation inactivation in aqueous phase.

Hyperlipidemia and type I 5'-monodeiodinase activity: regulation by selenium supplementation in rabbits.

Male New Zealand White rabbits were divided into three groups: (I) control, (II) high-fat-diet (HFD) fed, and (III) HFD fed + selenium supplemented. After 3 mo of treatment, there was a significant increase in serum cholesterol and triglycerides in the HFD-fed group as compared to the control. However in the selenium (Se)-supplemented group, the levels of serum cholesterol and triglycerides were significantly less as compared to group II. HFD feeding resulted in decreased serum Se levels, but supplementation of dietary Se along with HFD, as in group III, showed an apparent increase in its levels. The Se-dependent glutathione peroxidase (GSH-Px) activity in the liver and the aorta increased significantly in HFD-fed animals and also showed an additional significant increase on Se supplementation. Both serum T3 and T4 levels showed a significant decrease on HFD feeding. However, supplementation of Se led to a significant increase in the levels of these parameters viz-à-viz HFD-fed animals. HFD feeding significantly decreased the activity of type I iodothyronine 5'-deiodinase (5'-DI) in the liver from group II rats. On supplementation of Se along with HFD, the activity increased in the liver. However, there was no significant change in its activity in the aorta. The 5'-DI activity in the thyroid showed an opposite trend in comparison with peripheral tissues (i.e., liver). The important finding of this study is that in the hyperlipidemic state, deiodinase in the thyroid behaves in a different manner as compared to its activity in extrathyroidal tissues.

Selenium supplementation and diet induced hypercholesterolemia in the rat: changes in lipid levels, malonyldialdehyde production and the nitric oxide synthase activity.

Male Sprague Dawley rats were divided into three groups, viz (I) Controls, (II) High fat diet (HFD) fed, (III) HFD fed+selenium supplemented. After three months of treatment, there were significant increases in serum cholesterol and triglycerides in HFD fed group as compared to control. However, in Se supplemented group, the levels of serum cholesterol and triglycerides were significantly less as compared to group II. Selenium-dependent glutathione peroxidase (GSH-Px) activity in the liver and the aorta increased significantly in HFD fed animals and also showed additional significant increase on selenium supplementation. Malonyldialdehyde (MDA) concentrations in serum, liver and aorta and the activity of nitric oxide synthase (NOS; evident from reactive nitrogen intermediates and citrulline levels) in plasma showed significant increases in HFD fed group. However, supplementation of selenium led to a significant reduction in the levels of these parameters vis-a-vis HFD fed animals except in MDA levels in the serum and the liver where this decrease was non-significant. The important finding of this study is that selenium supplementation modulates the sequences favoring pathogenesis of atherosclerosis.

LDH-C4-substrate binary complexes studied by intrinsic fluorescence method.

Lactate dehydrogenase-C4 (LDH-C4) has been studied in presence of substrates using intrinsic fluorescence measurements. Excitation maximum of LDH-C4 occurred at 282 nm whereas fluorescence emission maximum was obtained at 340 nm. Fluorescence intensities at 340 nm showed that ligands viz. NAD+, NADH, pyruvate and lactate quench the relative fluorescence intensities of LDH-C4 in a concentration dependent manner. NAD+ and NADH produced a maximum quenching between 92-93% while pyruvate and lactate quenched the fluorescence of LDH up to 29% and 21% respectively. Association constants (Ka) based on fluorescence measurements were 6.05 x 10(4)M-1, 20 x 10(4)M-1, 0.113 x 10(4)M-1 and 0.3 x 10(4)M-1, for NAD+, NADH, lactate and pyruvate respectively. Stern-Volmer constants (Ksv) show that NAD+ and NADH have single Ksv of 4.07 x 10(4)M-1 and 1.47 x 10(5)M-1, whereas lactate and pyruvate indicated quenching reaction to be made up of two components. Ksv at low and high concentration of lactate respectively were 0.645 x 10(2)M-1 and 0.05 x 10(2)M-1, whereas corresponding Ksv with pyruvate were 1.008 x 10(3)M-1 and 0.408 x 10(3)M-1. Low Ksv at higher concentrations suggested that the aromatic chromophores are located within a hydrophobic environment. Red shift in fluorescence maximum (lambda max) by 2nm with lactate and 6nm with pyruvate showed that interaction of these ligands with LDH-C4 exposes some buried chromophores of the enzyme to the surface.

Quenching of intrinsic fluorescence of sperm specific LDH by optical isomers of gossypol.

Intrinsic fluorescence of LDH-C4 has been studied in the presence of optical isomers of gossypol. The study showed that fluorescence due to tryptophan residues after excitation of LDH at 282 nm is quenched by each gossypol enantiomere in a concentration dependent manner. Half of the maximum quench (Q50%) of enzyme occurred with gossypol (-) at 0.9 x 10(-4) mol/l and with gossypol (+) at 1.4 x 10(-4) mol/l showing a maximum quench (Qmax) of 45% and 65% respectively, with a corresponding association constant (Ka) of 1.0 x 10(4) l/mol and 0.4 x 10(4) l/mol. Stern-Volmer constant (Ksv) inferred that quenching of LDH comprises at least two components with two different Ksv values. Ksv(I) and Ksv(II) between LDH-C4 and gossypol (-) were 1.97 x 10(3) l/mol and 1.22 x 10(3) l/mol, and those between LDH-C4 and gossypol(+) were 2.3 x 10(3) l/mol and 1.56 x 10(3) l/mol. Smaller Ksv at higher concentrations of gossypol indicated that some of the tryptophan residues in LDH-C4 are deeply buried within a hydrophobic environment. There was no blue or red shift of LDH-C4 when interacting with either of the gossypol enantiomeres.

Effect of diet induced hypercholesterolemia and selenium supplementation on nitric oxide synthase activity.

The aim of the present study was to examine the activity of nitric oxide synthase (NOS, EC 1.14.23) in plasma of high fat diet (HFD, 2% cholesterol and 100 g table butter/kg diet) and HFD + selenium (Se, 1 ppm as sodium selenite) fed rabbits for three months. Significant increase in the serum cholesterol and triglyceride levels in HFD fed group was observed. The activity of NOS also increased very significantly. However in Se supplemented animals, there was a significant reduction in serum cholesterol as well as in plasma NOS activity relative to HFD fed animals. It is concluded that the protective effect of Se on HFD induced NOS activity acts probably through its antioxidant/inhibitory action.