Komagataella kurtzmanii sp. nov., a new sibling species of Komagataella (Pichia) pastoris based on multigene sequence analysis

A novel methanol assimilating yeast species Komagataella kurtzmanii is described using the type strain VKPM Y-727 (=KBP Y-2878 = UCD-FST 76-20 = Starmer #75-208.2 = CBS 12817 = NRRL Y-63667) isolated by W.T. Starmer from a fir flux in the Catalina Mountains, Southern AZ, USA. The new species is registered in MycoBank under MB 803919. The species was differentiated by divergence in gene sequences for D1/D2 LSU rRNA, ITS1-5.8S-ITS2, RNA polymerase subunit I, translation elongation factor-1α and mitochondrial small subunit rRNA. K. kurtzmanii differs from its phenotypically similar sibling species Komagataella pastoris, Komagataella pseudopastoris, Komagataella phaffii, Komagataella populi and Komagataella ulmi by absence of growth at 35 °C and inability to assimilate trehalose.     

Structural and functional study of Legionella pneumophila effector RavA

Legionella pneumophila is an intracellular pathogen that causes Legionnaire''s disease in humans. This bacterium can be found in freshwater environments as a free‐living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella‐containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER‐derived secretory vesicles and membranes forming the Legionella‐containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α‐helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C‐terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C‐terminal sequence C⁴⁰⁹WTSFCGLF⁴¹⁷. Yeast‐two‐hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.     

The Sperm Ultrastructure and Some Reproductive Characteristics of the Chemosymbiotic Bivalve Calyptogena pacifica Dall, 1891 (Vesicomyidae: Pliocardiinae)

Russian Journal of Marine Biology - Pliocardiines (Bivalvia: Vesicomyidae: Pliocardiinae) are a chemosymbiotrophic group of bivalve mollusks that are obligate for reducing environments. These...     

Peroxidase Mechanism of Lipid-dependent Cross-linking of Synuclein with Cytochrome c: PROTECTION AGAINST APOPTOSIS VERSUS DELAYED OXIDATIVE STRESS IN PARKINSON DISEASE*

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that α-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H₂O₂. Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.Lewy bodies (LBs),3 mitochondrial impairment, and oxidative stress are cardinal features of Parkinson disease (PD) and several related neurodegenerative disorders (1, 2). Aggregation of α-synuclein (Syn), an abundant protein in synaptic terminals, plays a major role in the formation of LBs (3, 4). Neither the mechanisms of LB production nor their pathogenic or protective roles in neurodegeneration are well understood.In nigrostriatal dopaminergic synaptic terminals, mitochondria, harboring a host of death-initiating factors, are in peril of damage by reactive oxygen species generated by disrupted electron transport and/or oxidative metabolism of dopamine (DA). Because cytochrome c (cyt c)-dependent formation of apoptosomes and activation of caspases designates a point of no return in the apoptotic program, release of proapoptotic factors from synaptic mitochondria could threaten the integrity of the entire neuron. How neurons protect themselves against inadvertent release of death signals from damaged synaptic mitochondria is not known.The N-terminal fragment of Syn contains six variants of an 11-amino acid consensus motif that include an apolipoprotein-like class A2 helix participating in binding of different lipids, particularly anionic phospholipids (5). This domain is believed to be important for Syn functions in regulation of neuronal lipid metabolism, particularly turnover of a mitochondria-specific phospholipid, cardiolipin (CL) (6). However, the relevance of the Syn lipid binding capacity in regulating neuronal injury (apoptotic) responses has not been established.It is believed that oxidative stress participates in the accumulation of LB and Lewy neurites (LN) through yet to be identified pathways (7). Reportedly, Syn is co-localized with cyt c in LBs (8), indicating a potential interaction between the two proteins. Because cyt c is a redox-active hemeprotein (9, 10), its presence in the LBs in conjunction with Syn may also provide a mechanistic link of LBs with oxidative stress. We have recently reported that cyt c interacts with CL in mitochondria early in apoptosis and with phosphatidylserine (PS) in the plasma membrane after its release into the cytosol (11, 12). In both cases, this results in redox activation of cyt c and the production of complexes with high peroxidase activity that effectively catalyze peroxidation of the respective phospholipids (13).Based on these facts, we hypothesize and provide experimental evidence that Syn acts as a sacrificial scavenger of cytosolic cyt c inadvertently released from synaptic mitochondria to prevent its migration into the soma, i.e. spread of the proapoptotic signal and cell death. This vital function is realized through the emergence of a peroxidase activity of the cyt c-Syn-phospholipid complex that cross-links its components and yields covalently conjugated protein-lipid hetero-oligomers. The latter maintain lingering peroxidase activity. Thus protection against acute apoptotic cell death comes with a penalty of Syn-cyt c aggregation into a peroxidase complex capable of inducing protracted oxidative stress. Our results present a novel biochemical mechanism likely involved in Lewy body formation and explain a known paradox of a dual protective and deleterious role that Syn plays in neuronal cells.     

Morphology of gametes of mollusks, echinoderms, and brachiopods in systematics and phylogeny

The morphology of eggs and sperm of echinoderms, mollusks, and brachiopods was studied and compared. The gametes of inarticulate brachiopods (two classes Lingulata and Craniata and two subphyla Linguliformea and Craniaformea) are shown to have significant morphological differences from those of articulate brachiopods (extant class Rhynchonellata, subphylum Rhynchonelliformea). Inarticulate brachiopods have similar sperm morphology to that of primitive brachiopods, bivalves and some polychaetes that have external fertilization. Sperm morphology of articulate brachiopods is similar to that of echinoderms, which are considered to be typical deuterostomate invertebrates. This similarity supports an early deviation of lophophore-bearing animals from Bilateria, before this lineage branched into Protostomia and Deuterostomia. Similar gamete morphology in Lingulata and Craniata supports the view that inarticulate brachiopods should be retained as a supraclass taxon for comparison with other Lophotrochozoa, in particular with phoronids, bryozoans, and mollusks. Based on the new data on the gamete morphology in inarticulate brachiopods, we propose the name Lingulophyles with the type genus Lingula, and for articulate brachiopods Coptothyrophyles with the type genus Coptothyris.     

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane,by ascorbate/dihydrolipoate in living HL-60 cells

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

A simulation model of <Emphasis Type="Italic">Escherichia coli</Emphasis> osmoregulatory switch using E-CELL system

Background  

Bacterial signal transduction mechanism referred to as a "two component regulatory systems" contributes to the overall adaptability of the bacteria by regulating the gene expression. Osmoregulation is one of the well-studied two component regulatory systems comprising of the sensor, EnvZ and the cognate response regulator, OmpR, which together control the expression of OmpC and OmpF porins in response to the osmolyte concentration.     

A steady state analysis indicates that negative feedback regulation of PTP1B by Akt elicits bistability in insulin-stimulated GLUT4 translocation

Background  

The phenomenon of switch-like response to graded input signal is the theme involved in various signaling pathways in living systems. Positive feedback loops or double negative feedback loops embedded with nonlinearity exhibit these switch-like bistable responses. Such feedback regulations exist in insulin signaling pathway as well.