Six structurally related 3-keto-substituted macrolide antibiotics (ketolides) were compared for concentration-dependent inhibitory effects on growth rate, viable cell number, and protein synthesis rates in Staphylococcus aureus cells. Inhibitory effects on 50S ribosomal subunit formation were also examined, as this is a second target for these antibiotics. A concentration range of 0.01 to 0.1 μg/ml was tested. An IC50 for inhibition of translation and 50S synthesis was measured for each compound, to relate structural features to inhibitory activity. ABT-773 was the most effective of the six compounds tested with an IC50 = 0.035 μg/ml. HMR 3004 was almost as effective with an IC50 = 0.05 μg/ml. Two 2-fluoroketolides (HMR 3562 and HMR 3787) were equivalent in their inhibitory activity with an IC50 = 0.06 μg/ml. Telithromycin (HMR 3647) had an IC50 = 0.08 μg/ml, and HMR 3832 was least effective with an IC50 = 0.11 μg/ml. Each antibiotic had an equivalent inhibitory effect on translation and 50S subunit formation. These results indicate specific structural features of these antimicrobial agents, which contribute to defined inhibitory activities against susceptible organisms.

The effects of erythromycin on the formation of ribosomal subunits were examined in wild-type Escherichia coli cells and in an RNase E mutant strain. Pulse-chase labelling kinetics revealed a reduced rate of 50S subunit formation in both strains compared with 30S synthesis, which was unaffected by the antibiotic. Growth of cells in the presence of [14C]-erythromycin showed drug binding to 50S particles and to a 50S subunit precursor sedimenting at about 30S in sucrose gradients. Antibiotic binding to the precursor correlated with the decline in 50S formation in both strains. Erythromycin binding to the precursor showed the same 1:1 stoichiometry as binding to the 50S particle. Gel electrophoresis of rRNA from antibiotic-treated organisms revealed the presence of both 23S and 5S rRNAs in the 30S region of sucrose gradients. Hybridization with a 23S rRNA-specific probe confirmed the presence of this species of rRNA in the precursor. Eighteen 50S ribosomal proteins were associated with the precursor particle. A model is presented to account for erythromycin inhibition of 50S formation.

Ketolides are the most recent generation of antimicrobials derived from the 14-membered ring macrolide, erythromycin A. The main structural feature that differentiates ketolides from erythromycin is the keto group, which replaces the L-cladinose moiety at position 3 of the macrolactone ring. The keto group bestows greater acid stability on the drugs, and enables them to bind to their ribosomal target without causing expression of MLSB resistance in inducible strains. Several ketolides are described here, including ABT 773 and telithromycin (HMR 3647), both of which possess a carbamate at C11/C12 of the macrolactone ring. In telithromycin, which is the first ketolide to be approved for clinical use, the carbamate is linked to an alkyl-aryl extension, which is responsible for the increased potency of this compound relative to macrolides. This review examines how the structural differences between macrolides and the new ketolides are related to their antimicrobial activities in inhibiting protein synthesis and blocking the assembly of new ribosomal subunits.

The rationale for the present review is that oxysterols found in oxidized LDL (oxLDL) play a role in atherogenesis. This perspective is based on studies that show that induction of apoptosis in vascular cells is an important process in atherogenesis, that apoptosis can be induced by oxLDL, and that the oxysterol component of oxLDL is responsible for its proapoptotic activity. The evidence for these concepts is reviewed, as are studies on the mechanisms by which oxysterols can induce apoptosis. An elevation in intracellular calcium appears to be an early signal transduction event that leads to apoptosis through both the extrinsic and intrinsic apoptotic pathways.

The continuing increase in antibiotic-resistant pathogenic bacterial has stimulated research on the development of new antimicrobial agents and the identification of new cellular targets. One such target is the sequence of assembly steps required for the formation of bacterial ribosomal subunits. A large number of different protein synthesis inhibitors which affect large subunit function also prevent the 50S particle from being formed in growing cells. These compounds include the macrolide and ketolide antibiotics as well as certain lincosamides, B-type streptogramins and several other structurally unrelated translational inhibitors. This review describes the activities of these compounds as inhibitors of 50S subunit formation. For most of these drugs, their inhibitory effect on particle synthesis is equivalent to their effect on translation. This new target is thus of equal importance to translational inhibition as a mechanism of action of these compounds. Features of the 50S subunit precursor particle as a target for these drugs are described. Finally a model is presented which accounts for this activity and predicts certain features of the substrate for erythromycin methylase activity in inducible cells. Antibiotics which target subunit formation preferentially are predicted to be important bactericidal agents.

The Plethodon glutinosus complex is composed of 16 lineages that are thought to have conserved morphological characteristics and rapid rates of diversification. Typically, these lineages are recognized as species, but the monophyly of some has been questioned. Three lineages have distributions that converge in the state of Alabama: Plethodon glutinosus, P. grobmani, and P. Mississippi. If these species are present in the state and are reproductively isolated, then we expected to recover three monophyletic lineages. If these species are present in the state, but exhibit extensive introgression, then we expected to recover sets of private haplotypes associated with each species and sets of shared haplotypes among species. We sampled 40 specimens of slimy salamanders from throughout the state. Samples were analyzed using two genes, cytochrome b (n = 38) and RPL12 (n = 17). Additionally, we added 47 cytochrome b sequences for Alabama specimens of the three species available on GenBank to examine relationships of this larger sample. We failed to recover three monophyletic lineages within any estimated gene tree and failed to recover sets of private haplotypes. Instead, haplotype-network structure revealed a single metapopulation. We conclude that Alabama contains a single species of slimy salamander, Plethodon glutinosus, with complex genetic connectivity throughout the state.

Early stages of inflammation are characterized by extensive oxidative insult by recruited and activated neutrophils. Secretion of peroxidases, including the main enzyme, myeloperoxidase, leads to the generation of reactive oxygen species. We show that this oxidative insult leads to polyunsaturated fatty acid (eg, docosahexaenoate), oxidation, and accumulation of its product 2-(v-carboxyethyl)pyrrole (CEP), which, in turn, is capable of protein modifications. In vivo CEP is generated predominantly at the inflammatory sites in macrophage-rich areas. During thioglycollate-induced inflammation, neutralization of CEP adducts dramatically reduced macrophage accumulation in the inflamed peritoneal cavity while exhibiting no effect on the early recruitment of neutrophils, suggesting a role in the second wave of inflammation. CEP modifications were abundantly deposited along the path of neutrophils migrating through the 3-dimensional fibrin matrix in vitro. Neutrophil-mediated CEP formation was markedly inhibited by the myeloperoxidase inhibitor, 4-ABH, and significantly reduced in myeloperoxidase-deficient mice. On macrophages, CEP adducts were recognized by cell adhesion receptors, integrin aMb2 and aDb2. Macrophage migration through CEP-fibrin gel was dramatically augmented when compared with fibrin alone, and was reduced by b2-integrin deficiency. Thus, neutrophil-mediated oxidation of abundant polyunsaturated fatty acids leads to the transformation of existing proteins into stronger adhesive ligands for aMb2- and aDb2-dependent macrophage migration. The presence of a carboxyl group rather than a pyrrole moiety on these adducts, resembling characteristics of bacterial and/or immobilized ligands, is critical for recognition by macrophages. Therefore, specific oxidation-dependent modification of extracellular matrix, aided by neutrophils, promotes subsequent aMb2- and aDb2-mediated migration/retention of macrophages during inflammation.

Sodium bicarbonate (NaHCO3) slows the decline in kidney function in patients with chronic kidney disease (CKD), yet the mechanisms mediating this effect remain unclear. The Dahl salt-sensitive (SS) rat develops hypertension and progressive renal injury when fed a high salt diet; however, the effect of alkali loading on kidney injury has never been investigated in this model. We hypothesized that NaHCO3 protects from the development of renal injury in Dahl salt-sensitive rats via luminal alkalization which limits the formation of tubular casts, which are a prominent pathological feature in this model. To examine this hypothesis, we determined blood pressure and renal injury responses in Dahl SS rats drinking vehicle (0.1 M NaCl) or NaHCO3 (0.1 M) solutions as well as in Dahl SS rats lacking the voltage-gated proton channel (Hv1). We found that oral NaHCO3 reduced tubular NH4+ production, tubular cast formation, and interstitial fibrosis in rats fed a high salt diet for 2 weeks. This effect was independent of changes in blood pressure, glomerular injury, or proteinuria and did not associate with changes in renal inflammatory status. We found that null mutation of Hv1 also limited cast formation in Dahl SS rats independent of proteinuria or glomerular injury. As Hv1 is localized to the luminal membrane of TAL, our data suggest that alkalization of the luminal fluid within this segment limits cast formation in this model. Reduced cast formation, secondary to luminal alkalization within TAL segments may mediate some of the protective effects of alkali loading observed in CKD patients.

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from -granules of activated platelets. Based on its structure, PF4 was assigned to the CXC family of chemokines and has been shown to have numerous effects on myeloid leukocytes. However, the receptor for PF4 remains unknown. Here, we demonstrate that PF4 induces leukocyte responses through the integrin Mac-1 (M2, CD11b/CD18). Human neutrophils, monocytes, U937 monocytic and HEK293 cells expressing Mac-1 strongly adhered to immobilized PF4 in a concentration-dependent manner. The cell adhesion was partially blocked by anti-Mac-1 mAb and inhibition was enhanced when anti-Mac-1 antibodies were combined with glycosaminoglycans, suggesting that cell-surface proteoglycans act cooperatively with Mac-1. PF4 also induced Mac-1-dependent migration of human neutrophils and murine WT, but not Mac-1-deficient macrophages. Coating of Escherichia coli bacteria or latex beads with PF4 enhanced their phagocytosis by macrophages by 4-fold, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by WT, but not Mac-1-deficient macrophages. As determined by biolayer interferometry, PF4 directly bound the MI-domain, the major ligand-binding region of Mac-1, and this interaction was governed by a Kd of 1.3 0.2 M. Using the PF4-derived peptide library, synthetic peptides duplicating the MI-domain recognition sequences and recombinant mutant PF4 fragments, the binding sites for MI-domain were identified in the PF4 segments Cys12–Ser26 and Ala57–Ser70. These results identify PF4 as a ligand for the integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated through its interaction with Mac-1.

Atherosclerosis is a complex inflammatory process characterized by monocyte recruitment into the arterial wall, their differentiation into macrophages, and lipid accumulation. Because integrin αMβ 2 (CD11b/CD18) mediates multiple diverse functions of leukocytes, we examined its role in atherogenesis. αM -/- /ApoE -/- and ApoE -/- mice were fed a control or high fat diet for 3 or 16 wk to induce atherogenesis. Unexpectedly, αM deficiency accelerated development of atherosclerosis in female but not in male mice. The size of aortic root lesions was 3-4.5-fold larger in female αM -/- /ApoE -/- than in ApoE -/- mice. Monocyte and macrophage content within the lesions was increased 2.5-fold in female αM -/- /ApoE -/- mice due to enhanced proliferation. αMβ 2 elimination promoted gender-dependent foam cell formation due to enhanced uptake of cholesterol by αM -/- /ApoE -/- macrophages. This difference was attributed to enhanced expression of lipid uptake receptors, CD36 and scavenger receptor A1 (SR-A1), in female mice. Macrophages from female αM -/- /ApoE -/- mice showed dramatically reduced expression of FoxM1 transcription factor and estrogen receptors (ER) a and b. As their antagonists inhibited the effect of 17b-estradiol (E2), E2 decreased CD36, SR-A1, and foam cell formation in ApoE -/- macrophages in an ERa- and ERb-dependent manner. However, female αM -/- /ApoE -/- macrophages failed to respond to E 2 and maintained elevated CD36, SR-A1, and lipid accumulation. FoxM1 inhibition in ApoE -/- macrophages reduced ERs and enhanced CD36 and SR-A1 expression, whereas FoxM1 overexpression in αM -/- /ApoE -/- macrophages reversed their proatherogenic phenotype.We demonstrate a new, surprising atheroprotective role of αMβ 2 in female ApoE -/- mice. αMβ 2 maintains ER expression in macrophages and E 2 -dependent inhibition of foam cell formation.