Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study

Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by beta-adrenergic stimulation. The mobility of PLB on SDS-PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, the oligomeric structure of PLB must be determined in its native milieu, a lipid bilayer containing the Ca-ATPase. Here we have used fluorescence energy transfer (FET) to study the oligomeric structure of PLB in SDS and dioleoylphosphatidylcholine (DOPC) lipid bilayers reconstituted in the absence and presence of Ca-ATPase. PLB was labeled, specifically at Lys 3 in the cytoplasmic domain, with amine-reactive fluorescent donor/acceptor pairs. FET between donor- and acceptor-labeled subunits of PLB in SDS solution and DOPC lipid bilayers indicated the presence of PLB oligomers. The dependence of FET efficiency on the fraction of acceptor-labeled PLB in DOPC bilayers indicated that it is predominantly an oligomer having 9-11 subunits, with approximately 10% of the PLB as monomer, and the distance between dyes on adjacent PLB subunits is 0.9 +/- 0.1 nm. When labeled PLB was reconstituted with purified Ca-ATPase, FET indicated the depolymerization of PLB into smaller oligomers having an average of 5 subunits, with a concomitant increase in the fraction of monomer to 30-40% and a doubling of the intersubunit distance. We conclude that PLB exists primarily as an oligomer in membranes, and the Ca-ATPase affects the structure of this oligomer, but the Ca-ATPase binds preferentially to the monomer and/or small oligomers. These results suggest that the active inhibitory species of PLB is a monomer or an oligomer having fewer than 5 subunits.     

DNA abasic lesions in a different light: solution structure of an endogenous topoisomerase II poison

Topoisomerase II is the target for several anticancer drugs that "poison" the enzyme and convert it to a cellular toxin by increasing topoisomerase II-mediated DNA cleavage. In addition to these "exogenous topoisomerase II poisons," DNA lesions such as abasic sites act as "endogenous poisons" of the enzyme. Drugs and lesions are believed to stimulate DNA scission by altering the structure of the double helix within the cleavage site of the enzyme. However, the structural alterations that enhance cleavage are unknown. Since abasic sites are an intrinsic part of the genetic material, they represent an attractive model to assess DNA distortions that lead to altered topoisomerase II function. Therefore, the structure of a double-stranded dodecamer containing a tetrahydrofuran apurinic lesion at the +2 position of a topoisomerase II DNA cleavage site was determined by NMR spectroscopy. Three major features distinguished the apurinic structure ( = 0.095) from that of wild-type ( = 0.077). First, loss of base stacking at the lesion collapsed the major groove and reduced the distance between the two scissile phosphodiester bonds. Second, the apurinic lesion induced a bend that was centered about the topoisomerase II cleavage site. Third, the base immediately opposite the lesion was extrahelical and relocated to the minor groove. All of these structural alterations have the potential to influence interactions between topoisomerase II and its DNA substrate.     

Sensitivity, specificity, and predictive values of three Salmonella rapid detection kits using fresh and frozen poultry environmental samples versus those of standard plating

To reduce human exposure to Salmonella spp. in poultry products, broiler chicken flocks have been tested by culture methods. Since the standard techniques may take 3 to 5 days, rapid detection methods have been developed. In this study we tested the performance of three rapid tests originally developed for food samples by using environmental samples obtained from poultry houses. These rapid tests were Reveal, an enzyme-linked immunosorbent assay from Neogen Corp.; BIND, a bacterial ice nucleation detection method from Idetek Corp.; and a filter monitor method from Future Medical Technologies, Inc. For the standard culture, brilliant green with novabiocin and xylose-lysine-tergitol-4 agar were used for presumptive identification, and identities were confirmed by using poly-O antisera. Environmental samples were collected from farms belonging to an integrated poultry company prior to chick placement and 1 week before slaughter. Sensitivities, specificities, and predictive values with 95% confidence intervals were calculated. Statistical differences were determined by using McNemar's chi square test. The sensitivities of the different tests were not stable, varying widely between sample times, and were affected by freezing of the samples. All of the rapid tests had low sensitivities, which led to many false-negative results. All tests were able to detect Salmonella spp. at a concentration of 10 CFU/ml in at least one of four trials. The BIND and Reveal tests were simple to use with multiple samples and reduced laboratory time by up to 1 day. Based on our results, we do not recommend that any of these rapid tests, in their present state of development, be utilized with environmental samples collected with drag swabs.     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant from Tolerant (SIFT) classified 226 of 508 variants (44%) as "Intolerant." Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as "Probably or possibly damaging." Another 9-15% of the variants were classed as "Potentially intolerant or damaging." The results from the two algorithms are highly associated, with concordance in predicted impact observed for approximately 62% of the variants. Twenty-one to thirty-one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as "Tolerant" or "Benign." Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.     

The delivery of benzyl penicillin to Staphylococcus aureus biofilms by use of liposomes

The delivery of benzyl penicillin [penicillin G (pen-G)] encapsulated in cationic liposomes to a pen-G-sensitive strain of Staphylococcus aureus immobilized in biofilms has been investigated. The cationic liposomes prepared by extrusion (VETs, diameter approximately 140 nm) were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dimethylammonium ethane carbamoyl cholesterol (DC-chol) at a molar ratio of 1.0:0.49:0.43. This composition containing 22 mole% of the cationic lipid DC-chol has been found previously (Kim et al. Colloids Surfaces A 1999, 149, 561-570) to be optimum for adsorption of the liposomes on S. aureus biofilms. The effectiveness of the liposomes to deliver pen-G to the biofilms immobilized on microtitre plates was assessed from the rate of growth of the cells after exposure to the liposomal drug carrier relative to free pen-G at the same concentration. The time to reach maximum growth rate from biofilms was investigated as a function of overall drug concentration in a range 2.9 x 10- 3 mM to 1.09 mM and as a function of time of exposure to liposomal drug in a range 1.5 s to 2 h. Liposomal drug delivery was most effective relative to free drug at low overall drug concentrations and short times of exposure. The time to reach maximum growth rate from S. aureus biofilms could be extended by a factor of approximately 4 relative to free drug by the use of liposomally encapsulated pen-G. The results were supported by direct measurements of the distribution of pen-G between biofilm and supernatant which showed enhanced values relative to free drug and a transient preferential uptake of drug induced by the liposomes. The study demonstrates that for low drug concentrations and short exposure times liposomal drug delivery greatly enhances the effectiveness of pen-G for inhibiting the growth of bacterial biofilms of the potentially pathogenic bacterium Staphylococcus aureus.