Two-stage optimization process for formulation of chitosan microspheres

The objective of the present study was to optimize the concentration of a chitosan solution, stirring speed, and concentration of drugs having different aqueous solubility for the formulation of chitosan microspheres. Chitosan microspheres (unloaded and drug loaded) were prepared by the chemical denaturation method and were subjected to measurement of morphology, mean particle size, particle size distribution, percentage drug entrapment (PDE), drug loading, and drug release (in vitro). Morphology of the microspheres was dependent on the level of independent process parameters. While mean particle size of unloaded microspheres was found to undergo significant change with each increase in concentration of chitosan solution, the stirring rate was found to have a significant effect only at the lower level (ie, 2000 to 3000 rpm). Of importance, spherical unloaded microspheres were also obtained with a chitosan solution of concentration less than 1 mg/mL. Segregated unloaded microspheres with particle size in the range of 7 to 15 microm and mean particle size of 12.68 microm were obtained in the batch prepared by using a chitosan solution of 2 mg/mL concentration and stirring speed of 3000 rpm. The highest drug load ( microg drug/mg microspheres) was 50.63 and 13.84 for microspheres containing 5-fluorouracil and methotrexate, respectively. While the release of 5-fluorouracil followed Higuchi's square-root model, methotrexate released more slowly with a combination of first-order kinetics and Higuchi's square-root model. The formation of chitosan microspheres is helped by the use of differential stirring. While an increase in the concentration of water-soluble drug may help to increase PDE and drug load over a large concentration range, the effect is limited in case of water-insoluble drugs.     

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets: role of nitric oxide

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.     

Concurrent translocation of multiple polypeptide chains through the proteasomal degradation channel

The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.     

Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease

Humans and mice deficient in Fas, a tumor necrosis factor (TNF)-receptor family member, cannot induce apoptosis of autoreactive cells, and consequently develop progressive lymphoproliferative disorders and lupus-like autoimmune diseases. Previous studies have shown that short-term administrations of agonistic monoclonal antibodies against CD137, another TNF-receptor family member, activate T cells and induce rejection of allografts and established tumors. Here we report that treatment with an agonistic monoclonal antibody to CD137 (2A) blocks lymphadenopathy and spontaneous autoimmune diseases in Fas-deficient MRL/lpr mice, ultimately leading to their prolonged survival. Notably, 2A treatment rapidly augments IFN-gamma production, and induces the depletion of autoreactive B cells and abnormal double-negative T cells, possibly by increasing their apoptosis through Fas- and TNF receptor-independent mechanisms. This study demonstrates that agonistic monoclonal antibodies specific for costimulatory molecules can be used as novel therapeutic agents to delete autoreactive lymphocytes and block autoimmune disease progression.     

Differential requirement for the nonhelical tailpiece and the C terminus of the myosin rod in Caenorhabditis elegans muscle

Myosin heavy chain (MHC) is a large, multidomain protein important for both cellular structure and contraction. To examine the functional role of two C-terminal domains, the end of the coiled-coil rod and the nonhelical tailpiece, we have generated constructs in which residues within these domains are removed or mutated, and examined their behavior in Caenorhabditis elegans striated muscle. Genetic tests demonstrate that MHC lacking only tailpiece residues is competent to support the timely onset of embryonic contractions, and therefore viability, in animals lacking full-length MHC. Antibody staining experiments show that this truncated molecule localizes as wild type in early stages of development, but may be defective in processes important for thick filament organization later in embryogenesis. Ultrastructural analysis reveals thick filaments of normal morphology in disorganized arrangement, as well as occasional abnormal assemblages. In contrast, molecules in which the four terminal residues of the coiled coil are absent or mutated fail to rescue animals lacking endogenous MHC. Loss of these four residues is associated with delayed protein localization and delayed contractile function during early embryogenesis. Our results suggest that these two MHC domains, the rod and the tailpiece, are required for distinct steps during muscle development.     

Red blood cell membrane essential fatty acid metabolism in early psychotic patients following antipsychotic drug treatment

A role of indices of oxidative stress, oxidative injury, and abnormal membrane phospholipid, specifically the phospholipid essential polyunsaturated fatty acids (EPUFAs) metabolism has been suggested based on studies in separate groups of patients with or without medication. The current study investigated the relationship between these biochemical measures in first-episode psychotic patients (N=16) at baseline and after 6 months of antipsychotic treatment (N=5 each with risperidone and olanzapine) and compared them to matched normal subjects. The indices of oxidative stress included: antioxidant enzymes; superoxide dismutase, glutathione peroxidase and catalase; and the oxidative injury as the levels of plasma lipid peroxides. The key membrane EPUFA's been; linolenic acid, arachidonic acid, nervonic acid, docosapentaenoic acid and docosahexaenoic acid. Furthermore, the changes in these biochemical measures were correlated with clinical symptomatology. Data indicated that, at baseline, reduced levels of antioxidant enzymes were associated with increased plasma lipid peroxides and reduced membrane EPUFAs, particularly omega-3 fatty acids. Furthermore, these biochemical measures normalized after 6 months of antipsychotic treatment. Parallel-improved psychopathology indicated that membrane EPUFA status might be partly affected by oxidative damage, which together may contribute to the pathophysiology and thereby, psychopathology of schizophrenia. These data also support the augmentation of antipsychotic treatment by supplementation with a combination of antioxidants and omega-3 fatty acids.     

Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid

Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.     

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA.

Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.