Structural and Biochemical Characterization of Compounds Inhibiting Mycobacterium tuberculosis Pantothenate Kinase

Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.     

Possible Role of Metal(II) Octacyanomolybdate(IV) in Chemical Evolution: Interaction with Ribose Nucleotides

We have proposed that double metal cyanide compounds (DMCs) might have played vital roles as catalysts in chemical evolution and the origin of life. We have synthesized a series of metal octacyanomolybdates (MOCMos) and studied their interactions with ribose nucleotides. MOCMos have been shown to be effective adsorbents for 5′-ribonucleotides. The maximum adsorption level was found to be about 50 % at neutral pH under the conditions studied. The zinc(II) octacyanomolybdate(IV) showed larger adsorption compared to other MOCMos. The surface area seems to important parameter for the adsorption of nucleotides. The adsorption followed a Langmuir adsorption isotherms with an overall adsorption trends of the order of 5′-GMP > 5′-AMP > 5′-CMP > 5′-UMP. Purine nucleotides were adsorbed more strongly than pyrimidine nucleotides on all MOCMos possibly because of the additional binding afforded by the imidazole ring in purines. Infrared spectral studies of adsorption adducts indicate that adsorption takes place through interaction between adsorbate molecules and outer divalent ions of MOCMos.     

Purification and characterization of pectin lyase secreted by Aspergillus flavus MTCC 10938

An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the K _m and k _cat values of the enzyme were obtained as 1.7 mg/ml and 66 s^?1, respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.     

Patient Preferences Regarding Device Reuse and Potential of Devices for Reuse - A Study in a Veteran Population

BackgroundMany cardiac patients need and undergo device implants. Veterans’ preferences regarding post-mortem handling of devices are not known. Cardiac patients in low- and middle-income countries who need but cannot afford devices rely on donations. Charitable organizations have successfully provided devices for reuse to such patients.ObjectiveWe estimated the number of devices with potential for possible reuse in a veteran population.MethodsBetween January and December 2008, at a tertiary medical center, veterans with implanted cardiac devices were surveyed using a questionnaire for their preferences regarding post-mortem handling. One choice was donation to charity for reuse. Although altruistic, it is unclear what percent of such devices have reuse potential. Retrospective chart review of veterans who underwent device implants between 1992 and 2007 identified a cohort of patients with Implantable Cardiac Defibrillators (ICDs) who had died by April 31st 2009. In this cohort, ICDs implanted in the year preceding the patient’s death were counted as having reuse potential.Results94 of 97 veterans completed the survey. 56% were unaware of how devices are handled after death. The top three preferences for postmortem handling were: return to manufacturer, return to hospital and donation for reuse. 88% were willing to sign an advance device directive. Retrospective review identified 161 veterans who had received 301 ICDs. Of these, 77 ICDs (25%) had median reuse potential of 3.1 years.ConclusionIn a VA cohort of deceased patients a substantial proportion of devices had reuse potential. Further research is needed to direct health policy.     

RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics

Rho family GTPases regulate diverse processes in human melanoma ranging from tumor formation to metastasis and chemoresistance. In this study, a combination of in vitro and in vivo approaches was utilized to determine whether RHOJ, a CDC42 homologue that regulates melanoma chemoresistance, also controls melanoma migration. Depletion or overexpression of RHOJ altered cellular morphology, implicating a role for RHOJ in modulating actin cytoskeletal dynamics. RHOJ depletion inhibited melanoma cell migration and invasion in vitro and melanoma tumor growth and lymphatic spread in mice. Molecular studies revealed that RHOJ alters actin cytoskeletal dynamics by inducing the phosphorylation of LIMK, cofilin, and p41‐ARC (ARP2/3 complex subunit) in a PAK1‐dependent manner in vitro and in tumor xenografts. Taken together, these observations identify RHOJ as a melanoma linchpin determinant that regulates both actin cytoskeletal dynamics and chemoresistance by activating PAK1.