Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes.

Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (ßF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: ßF41Y; ßK66Y; ßF71Y; ßT84Y; ßF85Y; and ßL96Y. As with ßF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (ßT84Y, and ßF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in ßT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 µM ascorbate over a time period of 60 min increasing from 10% in ßF41Y to over 50% in ßT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.

Responses of skeletal muscle size and anabolism are reproducible with multiple periods of unloading/reloading.

Mechanical unloading has long been understood to contribute to rapid and substantial adaptations within skeletal muscle, most notably, muscle atrophy. Studies have often demonstrated that many of the alterations resulting from disuse are reversed with a reintroduction of load and have supported the concept of muscle plasticity. We hypothesized that adaptations during disuse and recovery were a repeatable/reproducible phenomenon, which we tested with repeated changes in mechanical load. Rats were assigned to one of the following five groups: animals undergoing one or two bouts of hindlimb unloading (28 days), with or without recovery (56 day), or control. Following the completion of their final time point, posterior crural muscles were studied. Muscle sizes were lower following 28 days of disuse but fully recovered with a 56-day reloading period, regardless of the number of disuse/recovery cycles. Mixed protein fractional synthesis rates consistently reflected mass and loading conditions (supported by anabolic signaling), whereas the myofibrillar protein synthesis response varied among muscles. Amino acid concentrations were assessed in the gastrocnemius free pool and did not correlate with muscle atrophy associated with mechanical unloading. Muscle collagen concentrations were higher following the second unloading period and remained elevated following 56 days of recovery. Anabolic responses to alterations in load are preserved throughout multiple perturbations, but repeated periods of unloading may cause additive strain to muscle structure (collagen). This study suggests that whereas mass and anabolism are reproducibly reflective of the loading environment, repeated exposure to unloading and/or reloading may impact the overall structural integrity of muscle. NEW & NOTEWORTHY Repeatability should be considered a component of skeletal muscle plasticity during atrophy and recovery. Muscle anabolism is equally affected during a first or second disuse bout and returns equally with adequate recovery. Elevated muscle collagen concentrations observed after the second unloading period suggest altered structural integrity with repeated disuse.

Synthesis and biological evaluation of 3,5-diaryl isoxazoline/isoxazole linked 2,3-dihydroquinazolinone hybrids as anticancer agents.

A series of new 3,5-diaryl isoxazoline/isoxazole linked 2,3-dihydro quinazolinone hybrids with different linker architectures have been designed and synthesized. These compounds have been evaluated for their anticancer activity. One of the compounds 4c amongst this series has shown promising anticancer activity. Further some detailed biological assays relating to the cell cycle aspects and tubulin depolymerization activity have been examined with a view to understand the mechanism of action of this conjugate.

Synthesis and biological evaluation of cinnamido linked pyrrolo[2,1-c][1,4]benzodiazepines as antimitotic agents.

A series of new cinnamido-pyrrolo[2,1-c][1,4]benzodiazepine conjugates (4a-d and 5a-d) and their dimers (6a-d) have been designed, synthesized and evaluated for their biological activity. The anticancer screening of compound 4a by the NCI exhibited significant GI50 values ranging from 68 to 732 nM against 53 of 59 human cancer cell lines tested. Compounds 5a-d and 6a-d have also shown remarkable cytotoxic activity with GI50 values <0.1 microM concentrations in a large number of cell lines. Interestingly, compounds 5b and 6b have been identified as a new class of inhibitors of tubulin polymerization and their action has been rationalized by the cell cycle arrest in G0 and G2/M phase.

Remarkable enhancement in the DNA-binding ability of C2-fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines and their anticancer potential.

C2-Fluoro substituted DC-81, and its dimers that comprise of two C2-fluoro substituted DC-81 subunits tethered to their C8-position through simple alkane spacers as well as piperazine moiety side-armed with symmetrical alkyloxy spacers have been designed and synthesized. These fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines have shown remarkable DNA-binding ability and most of them possess promising anticancer activity, having GI(50) values in micromolar to nanomolar concentration range. DNA thermal denaturation studies show that some of these compounds (14a-c and 15) increase the DeltaT(m) values in the range of 28.9-38 degrees C, and this is further confirmed by the restriction endonuclease studies. This study illustrates the importance of introducing fluoro substitution at the C2-position apart from the incorporation of a piperazine ring in between the alkyloxy linker for enhancement of the DNA-binding ability in comparison to DSB-120 and SJG-136 (DeltaT(m)=10.2 and 25.7 degrees C). Moreover, the variations in the DNA-binding ability with respect to fluoro substitution in this class of dimers has been investigated by molecular modeling studies. Some representative C2-fluoro substituted dimers (8a and 14a) have also exhibited significant anticancer activity in the 60 cancer cell line assay of the National Cancer Institute (NCI).