Post-translational Modifications near the Quinone Binding Site of Mammalian Complex I

Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N^G and ω-N^G′ nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.     

Association of Over-The-Counter Pharmaceutical Sales with Influenza-Like-Illnesses to Patient Volume in an Urgent Care Setting

We studied the association between OTC pharmaceutical sales and volume of patients with influenza-like-illnesses (ILI) at an urgent care center over one year. OTC pharmaceutical sales explain 36% of the variance in the patient volume, and each standard deviation increase is associated with 4.7 more patient visits to the urgent care center (p<0.0001). Cross-correlation function analysis demonstrated that OTC pharmaceutical sales are significantly associated with patient volume during non-flu season (p<0.0001), but only the sales of cough and cold (p<0.0001) and thermometer (p<0.0001) categories were significant during flu season with a lag of two and one days, respectively. Our study is the first study to demonstrate and measure the relationship between OTC pharmaceutical sales and urgent care center patient volume, and presents strong evidence that OTC sales predict urgent care center patient volume year round.     

Neuronal pathfinding

Isolated SNARE proteins have been shown to confer only partial specificity on the fusion of artificial liposomes that mimic cellular membranes, modifying the SNARE hypothesis of membrane fusion.     

Performance evaluation of QuantiBRITE phycoerythrin beads.

BACKGROUND: The performance of QuantiBRITE phycoerythrin (PE) beads to standardize quantitation in terms of antibodies bound per cell (ABC) was evaluated by measuring precision, variation across multiple instruments, and variation across time. METHODS: For CD4 quantitation, whole blood was stained with a two-color CD4 reagent using a no-wash/no-lyse format. For CD69 quantitation, whole blood was activated with either phorbol myristate acetate (PMA) or CD3 beads and then stained with a three-color CD69 reagent using a lyse-no-wash format. RESULTS: Across 20 normal donors, the mean CD4 ABC was 51,000. Within-assay precision on quantitation of CD4 ABC on T cells had a coefficient of variance (CV) of <1.0%. Across multiple flow cytometers, quantitation of CD4 ABC had a CV of <5.0%. Within-donor CV on CD4 ABC on 20 donors across 2 months ranged from 1.3% to 3.2%. Within-assay precision on quantitation of CD69 on T cells activated with either PMA or CD3 beads had a CV of <3.0%. Within-donor CV of CD69 ABC across 1 month ranged from 2% to 18% on PMA-activated samples and from 7% to 24% on CD3 bead-activated samples. CONCLUSIONS: Our results indicate that the QuantiBRITE PE beads provide a useful tool for standardized analysis across labs. When used in conjunction with 1:1 conjugates of PE-to-monoclonal antibody, the QuantiBRITE PE beads provide a simple yet robust means of quantitating expression levels in terms of ABC.     

Cell biology

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in cell biology.     

Molecular mechanical studies of base-pair opening in d(CGCGC):d(GCGCG), dG5·dC5, d(TATAT):d(ATATA), and dA5·dT5 in the B and Z forms of DNA

Molecular mechanical energy refinement of double-helical pentanucleotide tetra-phosphates, d(CGCGC):d(GCGCG), dG₅·dC₅, d(TATAT):d(ATATA), and dA₅ ·dT₅ geometries, are presented in order to examine the energy required to open the Nl(purine) …? N3(pyrimidine) distance (base-pair opening) of a Watson-Crick base pair from its normal value of 3 Å to a value of 6 Å. The structural consequences of forcing base-pair opening is sequence dependent. For both dA₅ ·dT₅ and d(TATAT):d(ATATA), forcing the Nl (AdeKN3 (Thy) distance of the central base pair to a value of 6 Å slides the bases perpendicular to the helix axis forming a low-energy non-Watson-Crick base pair having an adenine amine hydrogen …? thymine carbonyl oxygen hydrogen bond. The two GC sequences behave differently from both AT sequences and differently from each other. Forcing the Nl(Gua) …? N3(Cyt) distance to 6 Å leads to unconventional structures in which hydrogen bonds are formed between the separated bases and the bases above or below them. These structures appear to be trapped in true local minima 6–10 kcal/mol higher in energy than the Watson-Crick structures. Preliminary simulations on d(CGCGC):d(GCGCG) in the Z geometry suggest the reason the Z form may be more refractory to proton exchange than the B form, consistent with experimental observations.     

Biodesulfurization of organic-sulfur compounds

A screening assay in which dibenzothiophene (DBT) or DBT-sulfone served as the only bioavailable source of sulfur was used to obtain two new bacterial isolates, strains UM9 and UM3, that desulfurized either substrate. Strain UM9 produced the desulfurized product, 2-hydroxybiphenyl (HBP); no other identifiable desulfurized products or released sulfate or sulfite were detected. Biodesulfurization activity occurred only for growing cultures and was depressed by free sulfate. Neither isolate grew on DBT, DBT-sulfone, or HBP as sole carbon sources. Under optimized conditions of pH and temperature, strain UM9 exhibited up to 35% greater biodesulfurization of DBT-sulfone than did UM3, and both isolates also desulfurized several other organic-sulfur compounds. The kinetics and characteristics of biodesulfurization by either UM3 or UM9, tentatively identified as species ofRhodococcus, indicated mechanisms different from those reported in the literature for other bacteria.