Mutations in tumour suppressor genes,l(2)gl andl(2)gd,alter the expression ofwingless inDrosophila

To understand the roles of two well known tumour suppressor genes.l(2)gl andl(2)gd in normal imaginal disc development inDrosophila, we have initiated a study to examine effect of mulations of these genes on the expression of genes involved in the patterning of the imaginal discs. In this study we show that the expression ofwingless, theDrosophila orthologue of the mammalian oncogeneWnt, is affected in the imaginal discs ofl(2)gl ⁴ andl(2)gd ¹ mutant individuals. In the tumourous wing imaginal discs froml(2)gl mutant larvae, the pattern ofwingless expression was progressively disrupted with an increase in the area of expression, Tumourous wing imaginal discs froml(2)gd homozygous individuals exhibited progressive broadening and extension of the wingless expressing domains. We suggest thatl(2)gl andl(2)gd might be involved in regulating post embryonic expression ofWingless.     

A review on coronary artery disease,its risk factors,and therapeutics

Coronary artery disease (CAD) is one of the major cardiovascular diseases affecting the global human population. This disease has been proved to be the major cause of death in both the developed and developing countries. Lifestyle, environmental factors, and genetic factors pose as risk factors for the development of cardiovascular disease. The prevalence of risk factors among healthy individuals elucidates the probable occurrence of CAD in near future. Genome-wide association studies have suggested the association of chromosome 9p21.3 in the premature onset of CAD. The risk factors of CAD include diabetes mellitus, hypertension, smoking, hyperlipidemia, obesity, homocystinuria, and psychosocial stress. The eradication and management of CAD has been established through extensive studies and trials. Antiplatelet agents, nitrates, β-blockers, calcium antagonists, and ranolazine are some of the few therapeutic agents used for the relief of symptomatic angina associated with CAD.     

Coaxial lines for multiphase power distribution

A coaxial cable can be used to reduce the magnetic and electric fields that extend into environments in the vicinity of transmission lines and distribution lines and in-house or building wiring for power distribution systems. The use of the coaxial geometry may prove useful in cases where there are environmental concerns with respect to health effects and in cases where there is a need to run high-speed data communications in close proximity to power distribution systems. © 1996 Wiley-Liss, Inc.     

Effect of reactive oxygen species on the metabolism of tryptophan in rat brain: Influence of age

In an earlier study, oxidation of tryptophan hydroxylase was implicated as its affinity was decreased with aging in rat brain. To establish any potential link between its oxidative damage and aging, we have determined the activities of antioxidant enzymes in midbrain, pons and medulla of 2, 12 and 24 month old Fisher 344 BNF1 rats. The results obtained suggest that the activities of antioxidant enzymes varied considerably with age and brain regions studied. Activities of Cu/Zn superoxide dismutase and glutathione peroxidase were found to increase from 2 to 12 months and then decrease in 24 month old rats. However catalase activity decreased consistently with the age. A parallel increase in the carbonyl content was observed in these brain regions indicating the oxidation of proteins. Reactive oxygen species when included in the incubation mixture decreased the activity of tryptophan hydroxylase in a concentration dependent manner. The loss of tryptophan hydroxylase activity induced by hydrogen peroxide and superoxide anion was prevented by catalase. However superoxide dismutase did not provide such protection. Sulfhydryl agents, cysteine, glutathione and dithiothreitol partially prevented the loss of activity. These studies suggest an involvement of reactive oxygen species for sulfhydryl oxidation of tryptophan hydroxylase in aging.     

Effect of hydroxypropyl beta cyclodextrin complexation on aqueous solubility,stability, and corneal permeation of acyl ester prodrugs of ganciclovir

The purpose of the study was to investigate the effect of hydroxypropyl beta cyclodextrin (HPβCD) on aqueous solubility, stability, and in vitro corneal permeation of acyl ester prodrugs of ganciclovir (GCV). Aqueous solubility and stability of acyl ester prodrugs of Ganciclovir (GCV) were evaluated in pH 7.4 isotonic phosphate buffer solution (IPBS) in the presence and absence of HPβCD. Butyryl cholinesterase-mediated enzymatic hydrolysis of the GCV prodrugs was studied using various percentage w/v HPβCD. In vitro corneal permeation of GCV and its prodrugs (with and without 5% HPβCD) across isolated rabbit cornea was studied using side-by-side diffusion cells. HPβCD-prodrug complexation was of the A_L type with values for complexation constants ranging between 12 and 108 M⁻¹. Considerable improvement in chemical and enzymatic stability of the GCV prodrugs was observed in the presence of HPβCD. The stabilizing effect of HPβCD was found to depend on the degree of complexation and the degradation rate of prodrug within the complex. Five percent w/v HPβCD was found to enhance the corneal permeation of only the most lipophilic prodrug GCV dibutyrate (2.5-fold compared with 0% HPβCD). All other prodrugs showed little or no difference in transport in the presence of 5% w/v HPβCD. Agitation in the donor chamber largely influenced the transport kinetics of GCV dibutyrate across cornea. Results indicate the presence of an unstirred aqueous diffusion layer at the corneal surface that restricts the transport of the highly lipophilic GCV dibutyrate prodrug. HPβCD improves corneal permeation by solubilizing the hydrophobic prodrug and delivering it across the mucin layer at the corneal surface.     

Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery

The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance (¹H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98 ± 0.089 and 10.84 ± 0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by ¹H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35 ± 56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.KEY WORDS: back-of-the-eye, drug delivery, formulation, mixed nanomicelles, posterior, rabbits, rapamycin/sirolimus, retina/choroid, sclera, topical eye drops     

Novel Pentablock Copolymer-Based Nanoparticulate Systems for Sustained Protein Delivery

The design, synthesis, and application of novel biodegradable and biocompatible pentablock (PB) copolymers, i.e., polyglycolic acid-polycaprolactone-polyethylene glycol-polycaprolactone-polyglycolic acid (PGA-PCL-PEG-PCL-PGA) and polylactic acid-polycaprolactone-polyethylene glycol-polycaprolactone-polylactic acid (PLA-PCL-PEG-PCL-PLA) for sustained protein delivery, are reported. The PB copolymers can be engineered to generate sustained delivery of protein therapeutics to the posterior segment of the eye. PB copolymers with different block arrangements and molecular weights were synthesized by ring-opening polymerization and characterized by proton nuclear magnetic resonance (¹H-NMR), gel permeation chromatography (GPC), and X-ray diffraction (XRD) spectroscopy. Immunoglobulin G (IgG) was selected as a model protein due to its structural similarity to bevacizumab. The influence of polymer molecular weight, composition, and isomerism on formulation parameters such as entrapment efficiency, drug loading, and in vitro release profile was delineated. Crystallinity and molecular weight of copolymers exhibited a substantial effect on formulation parameters. A secondary structure of released IgG was confirmed by circular dichroism (CD) spectroscopy. In vitro cytotoxicity, cell viability, and biocompatibility studies performed on human retinal pigment epithelial cells (ARPE-19) and/or macrophage cell line (RAW 264.7) demonstrated PB copolymers to be excellent biomaterials. Novel PB polymers may be the answer to the unmet need of a sustained release protein formulation.KEY WORDS: block copolymers, controlled drug delivery, IgG, intravitreal, nanoparticles, ocular delivery, pentablock copolymers, posterior segment, protein therapeutics, sustained drug delivery     

In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method

Background

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.

Results

Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3±0.66)×10⁷ cells per gram of liver tissue with a viability of 82.3±3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.

Conclusion

We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals.