Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401-410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.     

Protective effect of curcumin on gamma-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on gamma-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The gamma-radiation at different doses (1, 2 and 4Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4Gy irradiation. Curcumin pretreatment (1, 5 and 10microg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1Gy irradiation all the concentrations of curcumin (1, 5 and 10microg/ml) significantly protected the lymphocytes from radiation damage. At 2Gy irradiation, 5 and 10microg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4Gy irradiation both 1 and 5microg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10microg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against gamma-radiation induced cellular damage.     

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.     

Hymenobacter swuensis sp. nov., a Gamma-Radiation-Resistant Bacteria Isolated from Mountain Soil

Gram stain-negative and non-motile bacteria, designated as DY53^T and DY43, were isolated from mountain soil in South Korea prior exposure with 5 kGy gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strains belonged to the family Cytophagaceae in the class Cytophagia. 16S rRNA gene sequence similarity of strains DY53^T and DY43 was 100 %. The highest degrees of sequence similarities of strains DY53^T and DY43 were found with Hymenobacter perfusus A1-12^T (98.8 %), Hymenobacter rigui WPCB131^T (98.5 %), H. yonginensis HMD1010^T (97.9 %), H. xinjiangensis X2-1g^T (96.6 %), and H. gelipurpurascens Txg1^T (96.5 %). The DNA G+C content of the novel strains DY53^T and DY43 were 59.5 mol%. Chemotaxonomic data revealed that strains possessed major fatty acids such as C_15:0 iso, C_15:0 anteiso, C_16:1 ω5c, summed feature 3 (16:1 ω7c/ω6c), summed feature 4 (17:1 anteiso B/iso I) and C_17:0 iso, and major polar lipid was phosphatidylethanolamine. The novel strains showed resistance to gamma radiation, with a D10 value (i.e., the dose required to reduce the bacterial population by tenfold) in excess of 5 kGy. Based on these data, strains DY53^T and DY43 should be classified as representing a novel species, for which the name Hymenobacter swuensis sp. nov. is proposed, with the type strain DY53^T (=KCTC 32018^T = JCM 18582^T) and DY43 (=KCTC 32010).     

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.     

Structural link between γ-aminobutyric acid type A (GABAA) receptor agonist binding site and inner β-sheet governs channel activation and allosteric drug modulation

Rapid opening and closing of pentameric ligand-gated ion channels (pLGICs) regulate information flow throughout the brain. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner β-sheet trigger channel activation. Homology modeling reveals that the β4-β5 linker physically connects the neurotransmitter binding site to the inner β-sheet. Inserting 1, 2, 4, and 8 glycines in this region of the GABA(A) receptor β-subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the α- and γ-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating.     

Combined use of six-well polystyrene plates and thin layer chromatography for rapid development of optimal plant cell culture processes: application to taxane production byTaxus sp.

TwoTaxus (T. chinensis andT. baccata) cell suspension cultures were used as a model system to demonstrate the similarities of biomass accumulation and secondary metabolite (taxane) production obtained from cultures in six-well polystyrene plates and glass shake flasks (25 ml and 125 ml). Interference from binding of taxanes in cell-free culture broth to the polystyrene plates was minimal with 85% of the paclitaxel (Taxol®) and 100% of baccatin and 10-deacetyl-7-xylosyl-taxol remaining in the medium after 24 h beyond which no further binding was observed. A simple thin layer chromatography (TLC) procedure with a chloroform: acentonitrile (4:1) solvent system on silica gel was developed to simultaneously test up to 17 cultures for taxane production. The combination of six-well plate technology for experimentation and TLC for rapid taxane analysis can greatly accelerate the establishment of conditions for an optimalTaxus plant-cell culture process for taxane production.Abbreviations TLC Thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - HPLC high pressure liquid chromotography - UV ultraviolet - Rf retention factor     

Human psychophysics: Functional interpretation for contrast sensitivity versus spatial frequency curve

The hypothesis that neural processing in the human visual pathways compensates for both optical degradation as well as noise contamination at the photoreceptor level is introduced and shown to be consistent with the high frequency portion of the contrast sensitivity function for threshold detection of sinusoidal gratings in addition to the suprathreshold phenomenon of matching sinusoidal gratings of different spatial frequencies. This offers a unifying interpretation for why, at threshold conditions, the high spatial frequency portion of the image is blurred as severely by the nervous system as it is by the optics (e.g. Campbell and Green, 1965) while in extreme suprathreshold conditions the nervous system effectively deblurs the image (e.g. Georgeson and sullivan, 1975; Kulikowski, 1976). These conclusions do not necessitate a highly specific form of visual processing such as Fourier channeling.This research was conducted at Yale University, Department of Ophthalmology and Visual Science, New Haven, Connecticut, USA, throughout which period A.W.S. was a John Simon Guggenheim fellow     

Production of recombinant proteins using multiple-copy gene integration in high-cell-density fermentations of Ralstonia eutropha

We have previously reported the development of a novel protein expression system based on Ralstonia eutropha. In this study we report on the influence of gene copynumber on recombinant protein expression in R. eutropha. We compare recombinant gene stability and expression levels of chromosomal integration with a plasmid-based expression system. Single, double, and triple copies of a gene encoding organophosphohydrolase (OPH), an enzyme prone to inclusion-body formation in E. coli, were integrated into the R. eutropha chromosome. A linear increase between the concentration of soluble, active OPH and gene copynumber was found. Using a triple-copy integrant, we were able to produce approximately 4.3 g/L of OPH in a high-cell-density fermentation. This represents the highest titer reported to date for this enzyme, and is approximately 30 times greater than expression levels reported in E. coli.