The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function

The 11th influenza A virus gene product is an 87-amino-acid protein provisionally named PB1-F2 (because it is encoded by an open reading frame overlapping the PB1 open reading frame). A significant fraction of PB1-F2 localizes to the inner mitochondrial membrane in influenza A virus-infected cells. PB1-F2 appears to enhance virus-induced cell death in a cell type-dependent manner. For the present communication we have identified and characterized a region near the COOH terminus of PB1-F2 that is necessary and sufficient for its inner mitochondrial membrane localization, as determined by transient expression of chimeric proteins consisting of elements of PB1-F2 genetically fused to enhanced green fluorescent protein (EGFP) in HeLa cells. Targeting of EGFP to mitochondria by this sequence resulted in the loss of the inner mitochondrial membrane potential, leading to cell death. The mitochondrial targeting sequence (MTS) is predicted to form a positively charged amphipathic alpha-helix and, as such, is similar to the MTS of the p13(II) protein of human T-cell leukemia virus type 1. We formally demonstrate the functional interchangeability of the two sequences for mitochondrial localization of PB1-F2. Mutation analysis of the putative amphipathic helix in the PB1-F2 reveals that replacement of five basic amino acids with Ala abolishes mitochondrial targeting, whereas mutation of two highly conserved Leu to Ala does not. These findings demonstrate that PB1-F2 possesses an MTS similar to other viral proteins and that this MTS, when fused to EGFP, is capable of independently compromising mitochondrial function and cellular viability.     

Experimental and numerical determination of odorant solubility in nasal and olfactory mucosa

Odorant deposition in the nasal and olfactory mucosas is dependent on a number of factors including local air/odorant flow distribution patterns, odorant mucosal solubility and odorant diffusive transport in the mucosa. Although many of these factors are difficult to measure, mucosal solubility in the bullfrog mucus has been experimentally determined for a few odorants. In the present study an experimental procedure was combined with computational fluid dynamic (CFD) techniques to further describe some of the factors that govern odorant mucosal deposition. The fraction of odorant absorbed by the nasal mucosa (eta) was experimentally determined for a number of odorants by measuring the concentration drop between odorant 'blown' into one nostril and that exiting the contralateral nostril while the subject performed a velopharyngeal closure. Odorant concentrations were measured with a photoionization detector. Odorants were delivered to the nostrils at flow rates of 3.33 and 10 l/min. The velopharyngeal closure nasal air/odorant flows were then simulated using CFD techniques in a 3-D anatomically accurate human nose modeland the mucosal odorant uptake was numerically calculated. The comparison between the numerical simulations and the experimental results lead to an estimation of the human mucosal odorant solubility and the mucosal effective diffusive transport resistance. The results of the study suggest that the increase in diffusive resistance of the mucosal layer over that of a thin layer of water seemed to be general and non-odorant-specific; however, the mucosa solubility was odorant specific and usually followed the trend that odorants with lower water solubility were more soluble in the mucosa than would be predicted from water solubility alone. The ability of this approach to model odorant movement in the nasal cavity was evaluated by comparison of the model output with known values of odorant mucosa solubility.     

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.     

Studies on isolated subcellular components of cat pancreas

Summary Transport of alanine was studied in isolated plasma membrane vesicles from cat pancreas using a rapid filtration technique. The uptake is osmotically sensitive and the kinetics ofl-alanine transport are biphasic showing a saturable and a nonsaturable component. The saturable component is seen only when a sodium gradient directed from the medium to the vesicular space is present. Under this condition an overshooting uptake ofl-but not ofd-alanine occurs. The Na⁺ gradient stimulated uptake ofl-alanine is inhibited byl-serine andl-leucine and stimulated when the membrane vesicles had been preloaded withl-alanine,l-serine orl-leucine.The ionophore monensin inhibits stimulation of uptake caused by a sodium gradient. In the presence of valinomycin or carbonyl cyanidep-trifluoromethoxyphenylhydrazone (CFCCP), the sodium-dependent transport is augmented in vesicles preloaded with K₂SO₄ or H⁺ ions (intravesicular pH 5.5), respectively. In the presence of different anions, the Na⁺-dependent transport is stimulated according to increasing anionic penetration through membranes (lipid solubility). We conclude that a sodium dependent electrogenic amino acid transport system is present in pancreatic plasma membranes.     

Occurrence of microbodies in the green algaBracteacoccus cinnabarinus grown heterotrophically

Summary A comparative study was made of the ultrastructure and abundance of microbodies in the green algaBracteacoccus cinnabarinus grown photoautotrophically and heterotrophically on a conventional culture medium containing sodium acetate, potassium acetate and glucose. Several changes were observed in the cells maintained under these conditions. Most noticeably, cells grown on acetate in both light and dark were packed with lipid bodies. Microbodies were found to be closely appressed to the lipid bodies in cells grown heterotrophically in the dark on sodium acetate and potassium acetate. The average number of microbody profiles per cell was in general threefold greater in cells grown on sodium acetate than those grown on potassium acetate. No microbodies were observed in cells maintained photoautotrophically on the three carbon sources or in cells maintained photoautotrophically on Bristol's inorganic medium alone. Cytochemical staining with 3,3-diaminobenzidine indicated the presence of catalase in the microbodies. The presence of microbodies suggests that the organelle may be performing functions similar to glyoxysomes in higher plants, namely the net conversion to succinate of acetyl CoA derived from lipid degredation. It is also apparent thatBracteacoccus can grow well as a heterotroph in the dark when acetate is included in the culture medium as a source of carbon.     

Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds

The polyenoic fatty acid isomerase from Propioniumbacterium acnes (PAI) was expressed in E. coli and biochemically characterized. PAI catalyzes the isomerization of a methylene-interrupted double bond system to a conjugated double bond system, creating (10E,12Z)-conjugated linoleic acid (CLA). PAI accepted a wide range of free polyunsaturated fatty acids as substrates ranging from 18:2 fatty acids to 22:6, converting them to fatty acids with two or three conjugated double bonds. For expression of PAI in yeast the PAI-sequence encoding 20 N-terminal amino acid residues was altered for optimal codon usage, yielding codon optimized PAI (coPAI). The percentage of 10,12-CLA of total esterified fatty acids was 8 times higher in yeast transformed with coPAI than in cells transformed with PAI. CLA was detected in amounts up to 5.7% of total free fatty acids in yeast transformed with coPAI but none was detected in yeast transformed with PAI. PAI or coPAI under the control of the constitutive CaMV 35S promoter or the seed-specific USP promoter was transformed into tobacco plants. CLA was only detected in seeds in coPAI-transgenic plants. The amount of CLA detected in esterified fatty acids was up to 0.3%, in free fatty acids up to 15%.     

Cloning and functional expression in Escherichia coli of a cDNA encoding cardenolide 16'-O-glucohydrolase from Digitalis lanata Ehrh

A clone of cardenolide 16'-O-glucohydrolase cDNA (CGH I) was obtained from Digitalis lanata which encodes a protein of 642 amino acids (calculated molecular mass 73.2 kDa). The amino acid sequence derived from CGH I showed high homology to a widely distributed family of beta-glucohydrolases (glycosyl hydrolases family 1). The recombinant CGH I protein produced in Escherichia coli had CGH I activity. CGH I mRNA was detected in leaves, flowers, stems and fruits of D. lanata.     

Quality and intensity differences of carvone enantiomers when tested separately and in mixtures

Subjects were able to detect quality differences between R-(—)-and S-(+)-carvone solutions when the solutions were equatedfor subjective intensity or concentration. When the two carvoneswere mixed, both qualities were perceived.     

Comparisons of the estimates of smell, taste and overall intensity in young and elderly people

Absolute magnitude estimates of the smell, taste and overallintensity were examined in young (18–21 years) and elderly(61–94 years) people. The odorant/tastant pairs were eithersolutions of almond extract or lime/sucrose. For each age groupestimates of overall intensity were less than the sum of theestimates of each stimulus pair, these ratios were not affectedby the age of the subjects. This occurred even though, on average,when compared with young and elderly people when the odorantwas lime and the tastant sucrose. Thus, the data suggest thataging does not affect the process by which overall intensityis determined.     

Two specific populations of GABAergic neurons originating from the medial and the caudal ganglionic eminences aid in proper navigation of callosal axons

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE‐ and CGE‐derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT‐PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE‐ and CGE‐derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 647–672, 2013