Radioimmunoassay of metallothionein in rabbit,rat, mouse,Chinese hamster,and human cells

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.     

Induction Cardiac Pacing: A New Approach

A new approach to induction cardiac pacing using a “skin tunnel” transformer has been developed and used in a patient. Initial results are encouraging. If the system proves reliable in the long term it may have important applications in the delivery of power to implanted artificial organs.     

Role of the methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular growth, and virulence

Growth of bacteria and fungi on fatty acid substrates requires the catabolic beta-oxidation cycle and the anaplerotic glyoxylate cycle. Propionyl-CoA generated by beta-oxidation of odd-chain fatty acids is metabolized via the methylcitrate cycle. Mycobacterium tuberculosis possesses homologues of methylcitrate synthase (MCS) and methylcitrate dehydratase (MCD) but not 2-methylisocitrate lyase (MCL). Although MCLs share limited homology with isocitrate lyases (ICLs) of the glyoxylate cycle, these enzymes are thought to be functionally non-overlapping. Previously we reported that the M. tuberculosis ICL isoforms 1 and 2 are jointly required for growth on fatty acids, in macrophages, and in mice. ICL-deficient bacteria could not grow on propionate, suggesting that in M. tuberculosis ICL1 and ICL2 might function as ICLs in the glyoxylate cycle and as MCLs in the methylcitrate cycle. Here we provide biochemical and genetic evidence supporting this interpretation. The role of the methylcitrate cycle in M. tuberculosis metabolism was further evaluated by constructing a mutant strain in which prpC (encoding MCS) and prpD (encoding MCD) were deleted. The DeltaprpDC strain could not grow on propionate media in vitro or in murine bone marrow-derived macrophages infected ex vivo; growth under these conditions was restored by complementation with a plasmid containing prpDC. Paradoxically, bacterial growth and persistence, and tissue pathology, were indistinguishable in mice infected with wild-type or DeltaprpDC bacteria.     

Identification and functional impact of homo-oligomers of the human proton-coupled folate transporter

The proton-coupled folate transporter (PCFT; SLC46A1) is a proton-folate symporter that is abundantly expressed in solid tumors and normal tissues, such as duodenum. The acidic pH optimum for PCFT is relevant to intestinal absorption of folates and could afford a means of selectively targeting tumors with novel cytotoxic antifolates. PCFT is a member of the major facilitator superfamily of transporters. Because major facilitator superfamily members exist as homo-oligomers, we tested this for PCFT because such structures could be significant to PCFT mechanism and regulation. By transiently expressing PCFT in reduced folate carrier- and PCFT-null HeLa (R1-11) cells and chemical cross-linking with 1,1-methanediyl bismethanethiosulfonate and Western blotting, PCFT species with molecular masses approximating those of the PCFT dimer and higher order oligomers were detected. Blue native polyacrylamide gel electrophoresis identified PCFT dimer, trimer, and tetramer forms. PCFT monomers with hemagglutinin and His(10) epitope tags were co-expressed in R1-11 cells, solubilized, and bound to nickel affinity columns, establishing their physical associations. Co-expressing YPet and ECFP*-tagged PCFT monomers enabled transport and fluorescence resonance energy transfer in plasma membranes of R1-11 cells. Combined wild-type (WT) and inactive mutant P425R PCFTs were targeted to the cell surface by surface biotinylation/Western blots and confocal microscopy and functionally exhibited a "dominant-positive" phenotype, implying positive cooperativity between PCFT monomers and functional rescue of mutant by WT PCFT. Our results demonstrate the existence of PCFT homo-oligomers and imply their functional and regulatory impact. Better understanding of these higher order PCFT structures may lead to therapeutic applications related to folate uptake in hereditary folate malabsorption, and delivery of PCFT-targeted chemotherapy drugs for cancer.     

Ischemic cardiac tissue conditioned media induced differentiation of human mesenchymal stem cells into early stage cardiomyocytes

Mesenchymal stem cells (MSCs) are multipotent, can be easily expanded in culture and hence are an attractive therapeutic tool for cardiac repair. MSCs have tremendous potential to transdifferentiate to cardiac lineage both in vitro and in vivo. The present study examined the differentiation capacity of conditioned media derived from ischemic cardiac tissue on human MSCs. Human Bone marrow-derived MSCs after due characterization by immunocytochemistry and flow cytometry for MSC specific markers were induced by culture media derived from ischemic (n = 13) and non-ischemic (n = 18) human cardiac tissue. Parallel cultures were treated with 5-azacytidine (5-azaC), a potent cardiomyogen. MSCs induced with ischemic conditioned media formed myotube like structures, expressed sarcomeric Troponin I, alpha myosin heavy chain proteins and were positive for cardiac specific markers (Nkx2.5, human atrial natriuretic peptide, myosin light chain-2a, GATA-4) as was observed in 5-azaC treated cells. However, uninduced MSCs as well as those induced with non-ischemic cardiac conditioned media still maintained the fibroblast morphology even after 3 weeks post-induction. Transmission electron microscopic studies of cardiomyocyte-like cells derived from MSCs revealed presence of sarcomeric bands but failed to show gap junctions and intercalated discs as of adult cardiomyocytes. These findings demonstrate that ischemic cardiac conditioned media induces morphological and molecular changes in MSCs with cardiac features, but at a primitive stage. Proteomics analysis of the ischemic conditioned media revealed differential expression of three relevant proteins (C-type lectin superfamily member 13, Testis-specific chromodomain protein Y2 and ADP/ATP translocase 1), whose exact role in cardiac regeneration needs further analysis.     

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.