Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.     

Liposomes of terbutaline sulphate: in vitro and in vivo studies

In vitro studies were conducted to understand the comparative drug diffusion pattern, across artificial membrane, of the drug and of the prepared liposomes of different liposomal membrane composition. In vivo studies were carried out to determine the extent and time-course of pulmonary tissue uptake of administered liposomes containing terbutaline sulphate(TER) on rat lungs. In vitro studies revealed that the drug released from the prepared liposomes obeys Higuchi's diffusion controlled model. Different loading doses and release patterns of drug from the liposomes can be obtained by altering the PC:CHOL ratio and incorporation of cholesterol was found to reduce permeability of the membrane. Similarly drug absorption in vivo in rat's lung following intratracheal instillation, prolonged over 12 hr by liposomal entrapment of TER. The findings of present investigation indicated that liposomally encapsulated TER can be used for pulmonary delivery for maximizing the therapeutic efficacy and reducing undesirable side effects.     

Upregulation of macrophage plasma membrane and nuclear phospholipase D activity on ligation of the alpha2-macroglobulin signaling receptor: involvement of heterotrimeric and monomeric G proteins

The effect of ligating the alpha2-macroglobulin signaling receptor (alpha2MSR) with receptor-recognized forms of alpha2M (alpha2M*) was studied with respect to phospholipase D (PLD) activity in murine macrophages, their plasma membranes, and nuclei. PLD activity in plasma membranes and nuclei increased linearly up to a ligand concentration of about 100 pM of either alpha2M* or a cloned and expressed receptor binding fragment (RBF). The RBF binding site mutant K1370A, which binds with high affinity to alpha2MSR, also increased nuclear PLD activity comparable to RBF and alpha2M*. Phorbol dibutyrate caused a two- to threefold stimulation of membrane and nuclear PLD activity, whereas PLD activity was nearly abolished by downregulation of protein kinase C; prior treatment with staurosporin, genestein, cyclosporin A, actinomycin D; or chelation of intracellular Ca2+. In permeabilized macrophages, isolated plasma membranes, and nuclei, GTP-gamma-S increased alpha2M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-beta-S. Incubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sARFII, greatly reduced alpha2M*-stimulated PLD activity in the presence of GTP-gamma-S. Preincubation of plasma membranes with GDP-beta-S prior to the addition of GTP-gamma-S and recombinant ARF1 significantly inhibited alpha2M*-stimulation of PLD activity. Nuclear PLD activity was maximally stimulated in the presence of both GTP-gamma-S and rARF1, whereas plasma membrane PLD activity was maximally stimulated in the presence of rARF1, GTP-gamma-S, RhoA, and ATP. In contrast, nuclear PLD activity was not affected by RhoA either alone or in combination with GTP-gamma-S or ATP.     

Chemical fingerprinting of Andrographis paniculata using HPLC, HPTLC and densitometry

An attempt has been made to develop a method by which to determine the chemical fingerprint of Andrographis paniculata (Acanthaceae). High-performance thin layer chromatography (HPTLC) was used to analyse hexane, chloroform, methanol and water extracts of leaves of A. paniculata. A computerised densitometer was applied to the two-dimensional spectrographic image analysis of the HPTLC plates. An HPLC equipped with a photodiode array detector was used for the analyses of these different extracts. The analyses showed that andrographolide and neoandrographolide are absent in the hexane extract but are present in greater amounts in the methanol extract as compared with the other extracts. These chromatograms may serve as a chemical fingerprint of the drug A. paniculata for quality control purposes and in the preparation of formulations based on the drug.     

Short-term resistance to diet-induced obesity in A/J mice is not associated with regulation of hypothalamic neuropeptides

To investigate the mechanisms underlying long-term resistance of the A/J mouse strain to diet-induced obesity, we studied, over a period of 4 wk, the expression of uncoupling proteins in brown adipose tissue and the expression of hypothalamic neuropeptides known to regulate energy homeostasis and then used microarray analysis to identify other potentially important hypothalamic peptides. Despite increased caloric intake after 2 days of high-fat feeding, body weights of A/J mice remained stable. On and after 1 wk of high-fat feeding, A/J mice adjusted their food intake to consume the same amount of calories as mice fed a low-fat diet; thus their body weight and insulin, corticosterone, free fatty acid, and glucose levels remained unchanged for 4 wk. We found no changes in hypothalamic expression of several orexigenic and/or anorexigenic neuropeptides known to play an important role in energy homeostasis for the duration of the study. Uncoupling protein-2 mRNA expression in brown adipose tissue, however, was significantly upregulated after 2 days of high-fat feeding and tended to remain elevated for the duration of the 4-wk study. Gene array analysis revealed that several genes are up- or downregulated in response to 2 days and 1 wk of high-fat feeding. Real-time PCR analysis confirmed that expression of the hypothalamic IL-1 pathway (IL-1beta, IL-1 type 1 and 2 receptors, and PPM1b/PP2C-beta, a molecule that has been implicated in the inhibition of transforming growth factor-beta-activated kinase-1-mediated IL-1 action) is altered after 2 days, but not 1 wk, of high-fat feeding. The role of additional molecules discovered by microarray analysis needs to be further explored in the future.     

Synthesis and biological activity of N-aryl-2-aminothiazoles: potent pan inhibitors of cyclin-dependent kinases

N-Aryl aminothiazoles 6-9 were prepared from 2-bromothiazole 5 and found to be CDK inhibitors. In cells they act as potent cytotoxic agents. Selectivity for CDK1, CDK2, and CDK4 was dependent of the nature of the N-aryl group and distinct from the CDK2 selective N-acyl analogues. The N-2-pyridyl analogues 7 and 19 showed pan CDK inhibitory activity. Elaborated analogues 19 and 23 exhibited anticancer activity in mice against P388 murine leukemia. The solid-state structure of 7 bound to CDK2 shows a similar binding mode to the N-acyl analogues.     

Pulmonary absorption of liposomal levonorgestrel

The purpose of these studies was to achieve desired bioavailability after pulmonary administration of Levonorgestrel (LN) and to provide prolonged effective concentration of the drug in plasma and to reduce reported side effects of orally administered drug. The plain drug suspension, physical mixture (plain drug with liposomal constituents), and drug-encapsulated liposomes containing 10 μg of drug were instilled intratracheally in rats. Similarly, 10-μg drug suspension (LO) was administered orally. The blood samples were withdrawn at specific time intervals and were subjected to LN analysis by spectrofluorimetric technique. The plasma drug concentration data of both the treatments were plotted, and pharmacokinetics data were calculated and compared with that of oral administration. Percentage relative bioavailability (F^*) of 97.6% 98.6%, and 109.9% were observed after pulmonary administration of plain drug formulation (LP1), physical mixture (plain drug along with constituents of liposomes [LP2], and liposomal (LP3) formulations of the drug, respectively. Following oral administration, C_max of 14.4±0.6 ng/mL was observed at 2.1±0.2 hours followed by subtherapeutic concentration beyond 30±0.2 hours, while after pulmonary administration of LP1, LP2, and LP3 formulations, C_max of 4.4±0.4 ng/mL, 4.2±0.5 ng/mL, and 4.4±0.6 ng/ML were observed at 6.0±0.2 hours, 7.0±0.2 hours, and 6.8±0.2 hours, respectively, followed by maintenance of effective plasma drug concentration up to 60±2 hours. These studies demonstrate superiority of pulmonary drug delivery with regards to maintenance of effective therapeutic concentration of the LN in the plasma over a period of 6 to 60 hours. Hence, the pulmonary delivery is expected to reduce frequency of dosing and systemic side effects associated with oral administration of LN.