BACKGROUNDApolipoprotein A5 (ApoA5) originally gained attention as a regulator of serum triglyceride concentrations through transgenic mouse studies. Our group recently developed the first assay to quantify serum ApoA5 protein concentrations and demonstrated that they are increased by administration of a potent peroxisome proliferator-activated receptor-alpha agonist.METHODSTo better characterize the circulating ApoA5, the protein was purified from human serum, and a definitive N-terminal protein sequence was obtained. In light of previous observations that ApoA5 was present in VLDL and not LDL, plasma infranatant and intermediate-density lipoprotein (IDL) were analyzed for ApoA5. Because the mature protein contains a single unpaired cysteine, ApoA5 in human serum was immunoprecipitated, and its migration pattern was examined via Western blotting under reducing and nonreducing conditions to determine whether the protein circulates as a disulfide-linked homodimer or heterodimer.RESULTSDefinitive N-terminal protein sequences obtained from ApoA5 purified from human serum indicated that cleavage of the signal peptide occurs in vivo at the predicted site. We found ApoA5 in VLDL, HDL, and chylomicrons but not in LDL, IDL, or plasma infranatant. Under both reducing and nonreducing conditions, ApoA5 migrated mainly as a single band with a relative molecular mass (Mr) of approximately 39,000, indicating that the protein exists in serum as a monomer and not as a disulfide-linked homodimer or heterodimer.CONCLUSIONSOur data help characterize ApoA5 by defining its lipoprotein particle distribution, by determining its N-terminal protein sequence, and by demonstrating that the mature protein circulates mainly as a monomer and not as a disulfide-linked homodimer or heterodimer.

Apolipoprotein A-V (apoA-V) first gained attention as a regulator of triglycerides through transgenic mouse studies. Furthermore, peroxisome proliferator-activated receptor alpha (PPARalpha) agonists such as fenofibrate increase apoA-V mRNA expression. Our group recently developed the first assay to quantitate serum apoA-V levels. Therefore, we sought to determine whether administration of a PPARalpha agonist would increase circulating apoA-V. Cynomolgus monkeys were dosed for 14 days with 0.3 mg/kg/day LY570977 L-lysine, a potent and selective PPARalpha agonist. Blood samples were drawn throughout the treatment period and after a 2 week washout. Administration of the PPARalpha agonist caused a 50% decrease in triglycerides that reversed at washout. Serum apoA-V concentrations increased 2-fold, correlated inversely with triglycerides, and were reversible at washout. The apoA-V/apoC-III ratio increased >2-fold, with this increase also reversible at washout. These data demonstrate for the first time that a PPARalpha agonist increases circulating apoA-V protein levels and the apoA-V/apoC-III ratio.

BACKGROUND:The recently discovered apolipoprotein A5 (ApoA5) is fast gaining attention as a key regulator of serum triglyceride concentrations. An ApoA5 mouse knock-out model produced an approximately fourfold increase in serum triglycerides, whereas a knock-in model with human ApoA5 produced 50-70% lower concentrations of mouse serum triglycerides. In addition, peroxisome proliferator-activated receptor-alpha agonists, which are used clinically to lower serum triglyceride concentrations, cause increased ApoA5 mRNA expression. Despite these compelling molecular biology data, relatively little is known about ApoA5 protein in human serum.METHODS:To better understand circulating concentrations and lipoprotein particle distribution of ApoA5, we expressed the recombinant human ApoA5 protein and raised antibodies against both the NH(2) and COOH termini.RESULTS:Using the above reagents, we demonstrate for the first time that ApoA5 is present in human serum, although at much lower concentrations than other apolipoproteins such as ApoA1. Using a dual-antibody sandwich ELISA that we developed, we observed ApoA5 concentrations in human serum ranging from 24 to 406 microg/L compared with approximately 1 g/L for ApoA1. We also examined the lipoprotein particle distribution of ApoA5 and found that ApoA5 was detectable in VLDL, HDL, and chylomicrons, but not LDL.CONCLUSIONS:These data demonstrate for the first time that ApoA5 is a secreted protein present in human serum and is associated with specific lipoprotein particles. In addition, our data indicate that the circulating concentration of human ApoA5 is very low compared with other apolipoproteins.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis.

To characterize induction of VanA resistance a plasmid was constructed in which the gene for firefly luciferase lucA was placed under the control of the promoter for the VanA resistance genes, the vanH promoter. This system afforded convenient quantitative measurement of induction of the VanA genes. Glycopeptide antibiotics and antibiotics representing 19 different mechanisms of action were evaluated for their ability to induce. Antibiotics that acted as inducers were all inhibitors of late steps of peptidoglycan synthesis. These included moenomycin, bacitracin, tunicamycin, ramoplanin and glycopeptides, but not penicillin or other beta-lactam antibiotics. Glycopeptide antibiotics were the most potent inducers. Both glycopeptides with little or no antimicrobial activity and semisynthetic glycopeptides active against VanA resistant enterococci were inducers. Overall, results suggest that an induction response may involve both an internal signal, such as precursor accumulation, and the glycopeptide molecule itself as a signal. The system may be useful as a screen for new antimicrobial agents.

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/K-m values for the two purified enzymes (3,400 M-1 s(-1) for S-PBP2x and 11.2 M-1 s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity; Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.

The preparation and biological evaluation of 7 beta-[[2-(phenyl)thiazol-4-yl]acetamido]cephalosporins and 7 beta-[[2-(pyridin-2-yl)thiazol-4-yl] acetamido] cephalosporins, 9a-o, substituted at the c-position with acetyloxymethyl, chlorine, hydrogen, and methyl are described. Hantzsch's thiazole synthesis is employed to provide thiazoleacetic acids 5a-e, subsequently followed by Morpho CDI-assisted amidation to complete the synthesis of target cephalosporins 9a-o. These compounds display activity selectively against Grampositive bacteria, but are inactive against most Gram-negative bacteria tested. Those with acetyloxymethyl at the 3-position, i.e., 9a, 9e, 9i, 9m, and 9o, exhibit activity with minimal inhibitory concentrations of 16 mu g/mL or lower against four strains of methicillin-resistant staphylococci, namely Staphylococcus aureus X400 and S13E and Staphylococcus epidermidis 270 and 222. Notably, 9a displays an activity profile similar to that of vancomycin regarding its spectrum and potency.

Dirithromycin is the 9-N,11-O-oxazine adduct formed from 9(S)-erythromycylamine and 2-(2-methoxyethoxy)acetaldehyde in which the methoxyethoxymethyl substituent on the oxazine ring possesses the R configuration. Epidirithromycin is its isomer in which the methoxyethoxymethyl substituent has the opposite (S) configuration. Both compounds readily epimerize in solution, reaching an equilibrium ratio of 85:15 in favor of dirithromycin, given sufficient time. The rate of interconversion is dependent upon pH, temperature, and solvent. An enriched sample of epidirithromycin (95% purity) was synthesized by condensing erythromycylamine and 2-(2-methoxyethoxy)acetaldehyde in diethyl ether as the reaction solvent, and the product was fully characterized by nuclear magnetic resonance spectroscopy and high-pressure liquid chromatographic (HPLC) analysis. Both oxazine derivatives readily hydrolyze to erythromycylamine, so all three compounds exhibit the same antibiotic activity in vitro. In order to determine whether dirithromycin itself possesses significant antimicrobial activity without initial hydrolysis to erythromycylmine, inhibition of cell-free ribosomal protein synthesis was measured under conditions which were adapted to minimize hydrolysis, as measured by analytical HPLC in parallel experiments. Under these particular conditions, inhibition of ribosomal protein synthesis by dirithromycin was < 10% of the value measured for erythromycylamine.

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.

Daptomycin inhibits the formation of UDP-N-acetylmuramyl-pentapeptide in Bacillus megaterium by inhibiting active transport of amino acids incorporated into the pentapeptide. The ability of daptomycin to inhibit active transport and peptidoglycan formation may be due to its ability to disrupt the transmembrane electrochemical gradient.

Daptomycin (LY146032) caused a calcium-dependent dissipation of the membrane potential (delta psi) in Staphylococcus aureus without noticeably affecting the chemical gradient (delta pH) across the membrane. The effect of daptomycin on membrane energization may account for many of the inhibitory effects on macromolecular biosyntheses and membrane function reported for this antibiotic. Our evidence indicates that the bactericidal activity of daptomycin is dependent on an available delta psi.

New semi-naphthaquinone antibiotics A80915A, B, C, and D were isolated from the fermented broth of Streptomyces aculeolatus A80915 (NRRL 18422). Factors A and C, present in both the broth filtrate and mycelial methanol extract, and factors B and D, found predominantly in the broth filtrate, were recovered by extraction with ethyl acetate. Purification of the individual factors was accomplished by preparative reverse phase high performance liquid chromatograph on C18 bonded silica supports. Factors A through D show antimicrobial activity against Gram-positive aerobic and anaerobic organisms in vitro. Mechanism of action studies demonstrated nearly complete inhibition of macromolecular biosynthesis (protein, RNA, DNA, and cell wall) by A80915 factors A through D. A less highly cyclized semi-naphthaquinone, A80915 factor G, was isolated from the broth of the strain fermented in an alternate medium.

LY146032, a cyclic lipopeptide antibiotic, is an inhibitor of cell wall peptidoglycan biosynthesis in gram-positive bacteria. Although LY146032 at relatively high concentrations inhibited the in vitro polymerization of UDP-linked sugar precursors, inhibition of cell wall formation in intact Staphylococcus aureus and Bacillus megaterium cells did not lead to the accumulation of UDP-N-acetyl-muramyl (MurNAc)-peptide(s). Experiments that measured formation of UDP-MurNAc-peptides revealed that LY146032 inhibited the formation of these nucleotide-linked intermediates. This antibiotic had a disruptive effect on membrane permeability as evidenced by the loss of intracellular potassium immediately after exposure to the drug. The lack of any major disruption of the phosphoenolpyruvate:sugar phosphotransferase system indicated that the membrane is not likely a lethal target for this antibiotic. The findings are consistent with a mechanism by which LY146032 inhibits the formation of precursor molecules utilized in peptidoglycan biosynthesis. The observed membrane effects likely result from transit of the inhibitor to its lethal target site.

A plasmid conferring resistance to the aminocyclitol antibiotic hygromycin B was isolated from Escherichia coli. The gene conferring resistance to this drug was cloned in pBR322, and the gene was localized to a fragment of ca. 1,510 base pairs. Resistance to hygromycin B is determined by an aminocyclitol phosphotransferase that modifies hygromycin B and structurally related antibiotics. The specific modification of hygromycin B is a phosphorylation of the hydroxyl on the 4 position of the cyclitol ring (hyosamine). The presence of the phosphotransferase in E. coli correlates with reduced accumulation of [14C]hygromycin B.

Aminoglycoside-2"-O-adenylyltransferase was inhibited by 7-hydroxytropolone. Inhibition was competitive with respect to the cosubstrate ATP and appeared to require the unique vicinal arrangement of oxygens found in 7-hydroxytropolone. Combinations of 7-hydroxytropolone plus the appropriate aminoglycoside substrates were active against resistant bacteria possessing the adenylyltransferase. No potentiation was observed against other aminoglycoside-resistant or -susceptible strains. The fact that the inhibition of an aminoglycoside-modifying enzyme overcomes the poor uptake of aminoglycosides in resistant strains points to the singular importance of the inactivating enzyme as a determinant of resistance.