Albumin microspheres as carriers for the antiarthritic drug celecoxib

The present study investigates the preparation of celecoxib-loaded albumin microspheres and the biodistribution of technetium-99m (^99mTc)-labeled celecoxib as well as its microspheres after intravenous administration. Microspheres were prepared using a natural polymer BSA using emulsification chemical cross-linking method. The prepared microspheres were characterized for entrapment efficiency, particle size, and in vitro drug release. Surface morphology was studied by scanning electron microscopy. Biodistribution studies were performed by radiolabeling celecoxib (CS) and its microspheres (CMS) using^99mTc and injecting arthritic rats intravenously. The geometric mean diameter of the microspheres was found to be 5.46 μm. In vitro release studies indicated that the microspheres sustained the release of the drug for }6 days. Radioactivity measured in different organs after intravenous administration of celecoxib solution showed a significant amount of radioactivity in the liver and spleen. In case of celecoxib-loaded microspheres, a significant amount of radioactivity accumulated in the lungs. No significant difference (P>.1) in the radioactivity was observed between the inflamed joint and the noninflamed joint following intravenous injection of^99mTc-CS. However, in case of the microspheres (CMS), the radioactivity present in the inflamed joint was 2.5-fold higher than in the noninflamed joint. The blood kinetic studies revealed that celecoxib-loaded albumin microspheres exhibited prolonged circulation than the celecoxib solution.     

Targeting of the c-Abl tyrosine kinase to mitochondria in the necrotic cell death response to oxidative stress

The ubiquitously expressed c-Abl tyrosine kinase is activated in the response of cells to genotoxic and oxidative stress. The present study demonstrates that reactive oxygen species (ROS) induce targeting of c-Abl to mitochondria. We show that ROS-induced localization of c-Abl to mitochondria is dependent on activation of protein kinase C (PKC)delta and the c-Abl kinase function. Targeting of c-Abl to mitochondria is associated with ROS-induced loss of mitochondrial transmembrane potential. The results also demonstrate that c-Abl is necessary for ROS-induced depletion of ATP and the activation of a necrosis-like cell death. These findings indicate that the c-Abl kinase targets to mitochondria in response to oxidative stress and thereby mediates mitochondrial dysfunction and cell death.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Directional genome walking using PCR

We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walker primers that were designed with partial degeneracy. The biotinylated primary PCR products were immobilized on streptavidin-linked paramagnetic beads. This step removed all nonspecific amplification products, and the purified template was used for the second PCR using a nested primer and the walker primer-2 to increase specificity. This technique is potentially useful for cloning promoter regions and has been successfully used to isolate 5'-flanking genomic regions of many cDNA clones previously isolated by us.     

Phosphorylation of Bcl-2 and Bax Proteins during Hypoxia in Newborn Piglets

Studies indicate that phosphorylated Bcl-2 cannot form a heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypothesis that graded hypoxia in cerebral tissue induces the phosphorylation of Bcl-2, thus altering the heterodimerization of Bcl-2 with Bax and subsequently leading to apoptosis. Anesthetized, ventilated newborn piglets were assigned to a normoxic and a graded hypoxic group. Cerebral cortical neuronal nuclei were isolated and immunoprecipitated; immune complexes were separated and reacted with Bcl-2 and Bax specific antibodies. The results show an increased level of serine/tyrosine phosphorylated Bcl-2 in nuclear membranes of hypoxic animals. The level of phosphorylated Bcl-2 protein increased linearly with decrease in tissue PCr. The level of phosphorylated Bax in the neuronal nuclear membranes was independent of cerebral tissue PCr. The data shows that during hypoxia, there is increased phosphorylation of Bcl-2, which may prevent its heterodimerization with Bax and lead to increased proapoptotic activity due to excess Bax in the hypoxic brain. Further increased phosphorylation of Bcl-2 may alter the Bcl-2/Bax-dependent antioxidant, lipid peroxidation and pore forming activity, as well as the regulation of intranuclear Ca²⁺ and caspase activation pathways. We speculate that increased phosphorylation of Bcl-2 in neuronal nuclear membranes is a potential mechanism of programmed cell death activation in the hypoxic brain.     

In vitro plantlet production system for Kaempferia galanga,a rare Indian medicinal herb

A rapid clonal propagation system for Kaempferia galanga (Zingiberaceae), a rare folk medicinal herb has been developed. Various concentrations of 6-benzyladenine (BA) and a range of auxins have been investigated for in vitro plantlet production, using rhizomes as explants. In vitro plantlet production has been achieved on 0.75 × Murashige and Skoog (MS) medium supplemented with 12 μM BA, 3 μM ∝-naphthaleneacetic acid (NAA) and 3% sucrose. The procedure ensures 13-fold rate of plantlet production every 4 weeks. Hardened plantlets produced normal storage roots as the parent plants. Around 1,000 plantlets have been produced successfully for field transfer. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cross-neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma

Cross-reactive epitopes (CREs) are similar epitopes on viruses that are recognized or neutralized by same antibodies. The S protein of SARS-CoV-2, similar to type I fusion proteins of viruses such as HIV-1 envelope (Env) and influenza hemagglutinin, is heavily glycosylated. Viral Env glycans, though host derived, are distinctly processed and thereby recognized or accommodated during antibody responses. In recent years, highly potent and/or broadly neutralizing human monoclonal antibodies (bnAbs) that are generated in chronic HIV-1 infections have been defined. These bnAbs exhibit atypical features such as extensive somatic hypermutations, long complementary determining region (CDR) lengths, tyrosine sulfation and presence of insertions/deletions, enabling them to effectively neutralize diverse HIV-1 viruses despite extensive variations within the core epitopes they recognize. As some of the HIV-1 bnAbs have evolved to recognize the dense viral glycans and cross-reactive epitopes (CREs), we assessed if these bnAbs cross-react with SARS-CoV-2. Several HIV-1 bnAbs showed cross-reactivity with SARS-CoV-2 while one HIV-1 CD4 binding site bnAb, N6, neutralized SARS-CoV-2. Furthermore, neutralizing plasma antibodies of chronically HIV-1 infected children showed cross neutralizing activity against SARS-CoV-2 pseudoviruses. Collectively, our observations suggest that human monoclonal antibodies tolerating extensive epitope variability can be leveraged to neutralize pathogens with related antigenic profile.     

Rationalisation of regimens of treatment of kala-azar with sodium stibogluconate in India: a randomised study

The efficacy and safety of six regimens of treatment for kala-azar (visceral leishmaniasis) with sodium stibogluconate were evaluated in a prospective randomised study to ascertain the optimal treatment for Indian patients. Altogether 371 patients with kala-azar were randomised to receive sodium stibogluconate intramuscularly at a dose of 10 mg/kg body weight/day for 20 or 40 days (groups A and A1, respectively), 15 mg/kg body weight/day for 20 or 40 days (groups B and B1, respectively), or 20 mg/kg body weight/day for 20 or 40 days (groups C and C1, respectively). Patients were examined blind before and at the end of treatment and every month for six months. The number of patients who were apparently cured—that is, those whose temperature had returned to normal at the end of their regimen of treatment—was 45 (78%) in group A, 53 (87%) in group A1, 50 (81%) in group B, 60 (95%) in group B1, 58 (92%) in group C, and 62 (97%) in group C1.At six months 62 patients (97%) in group C1, 51 (81%) in group C, 54 (86%) in group B1, 42 (68%) in group B, 45 (74%) in group A1, and 33 (57%) in group A had not relapsed and were cured as confirmed by a bone marrow aspirate free of parasites. The differences between groups C1 and C, B1 and B, and A1 and A were significant. Logistic regression of the proportion cured with the dose and length of treatment showed that both factors were significant in improving the rate of cure; the highest dose for the longer time (group C1) had the best rate of cure. One patient in group C1, 12 in group C, nine in group B1, 18 in group B, 15 in group A1, and 23 in group A were cured with extended courses of 20 mg sodium stibogluconate. One patient in each of groups C1, B, A1, and A became unresponsive to antimony and were cured with pentamidine. One patient in each of groups C1, B, and A became unresponsive to both antimony and pentamidine. The patients tolerated the longer duration of treatment safely, and side effects were minor.Sodium stibogluconate should be given intramuscularly in the dosage of 20 mg/kg for at least 40 days, when patients should be assessed for further treatment if necessary. Such a regimen should achieve the highest rate of cure with low toxicity and low rates of relapse and unresponsiveness.