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Retapamulin inhibited protein biosynthesis and cell viability in methicillin-sensitive and methicillin-resistant Staphylococcus aureus organisms. A specific inhibitory effect on 50S ribosomal subunit formation was also found. Pulse-chase labeling experiments confirmed the specific inhibition of 50S subunit biogenesis. Turnover of 23S rRNA was found, with no effect on 16S rRNA amounts.

Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.

Intracellular recordings were made in vitro from guinea-pig cardiac ganglia to determine whether endogenous neuropeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) or substance P released during tetanic neural stimulation modulate cardiac neurone excitability and/or contribute to slow excitatory postsynaptic potentials (sEPSPs). When nicotinic and muscarinic receptors were blocked by hexamethonium and atropine, 20 Hz stimulation for 10 s initiated a sEPSP in all innervated neurones. In 40% of the cells, excitability was enhanced after termination of the sEPSP. This suggested that non-cholinergic receptor-mediated mechanisms contributed to the sEPSP and modulated neuronal excitability. Exogenous PACAP and substance P initiated a slow depolarization in the neurones whereas neuronal excitability was only increased by PACAP. When ganglia were treated with the PAC1 antagonist PACAP6-38 (500 nm), the sEPSP evoked by 20 Hz stimulation was reduced by ∼50% and an enhanced excitability occurred in only 10% of the cells. These observations suggested that PACAP released from preganglionic nerve terminals during tetanic stimulation enhanced neuronal excitability and evoked sEPSPs. After addition of 1 n m PACAP to the bath, 7 of 9 neurones exhibited a tonic firing pattern whereas in untreated preparations, the neurons had a phasic firing pattern. PACAP6-38 (500 nm) diminished the increase in excitability caused by 1 nm PACAP so that only 4 of 13 neurones exhibited a tonic firing pattern and the other 9 cells retained a phasic firing pattern. These findings indicate that PACAP can be released by tetanic neural stimulation in vitro and increase the excitability of intrinsic cardiac neurones. We hypothesize that in vivo PACAP released during preganglionic firing may modulate neurotransmission within the intrinsic cardiac ganglia.

Regulation of basal and cytokine (IL-6 and IL-1β)-induced expression of C-reactive protein (CRP) in human hepatoma Hep3B cells occurs during transcription. A critical transcriptional regulatory element on the CRP promoter is a C/EBP binding site overlapping a NF-κB p50 binding site. In response to IL-6, C/EBPβ and p50 occupy the C/EBP-p50 site on the CRP promoter. The aim of this study was to identify the transcription factors occupying the C/EBP-p50 site in the absence of C/EBPβ. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPβ. Such treated nuclei contain only C/EBPζ (also known as CHOP10 and GADD153) because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBPζ. EMSA using this extract revealed formation of a C/EBPζ-containing complex at the C/EBP-p50 site on the CRP promoter. This complex also contained RBP-Jκ, a transcription factor known to interact with κB sites. RBP-Jκ was required for the formation of C/EBPζ-containing complex. The RBP-Jκ-dependent C/EBPζ-containing complexes were formed at the C/EBP-p50 site on the CRP promoter in the nuclei of primary human hepatocytes also. In luciferase transactivation assays, overexpressed C/EBPζ abolished both C/EBPβ-induced and (IL-6 + IL-1β)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBPζ occupies the C/EBP site, an action that requires RBP-Jκ. Under induced conditions, C/EBPζ is replaced by C/EBPβ and p50. We conclude that the switch between C/EBPβ and C/EBPζ participates in regulating CRP transcription. This process uses a novel phenomenon, that is, the incorporation of RBP-Jκ into C/EBPζ complexes solely to support the binding of C/EBPζ to the C/EBP site.

Cyclodextrins (CDs) are one of the most successful solutions to the problem of poor drug solubility. In this study, we examined the in-vitro effects of three CDs on the solubility of letrozole, a breast cancer drug that is practically insoluble in water. The most promising, hydroxybutenyl-β- cyclodextrin (HBenβCD), was used for in-vivo studies in male and female Sprague-Dawley rats. Letrozole is a drug with dramatic gender-based differences in pharmacokinetics. For example, the terminal half-life (t1/2) of letrozole following intravenous administration in male rats was 11.5 ± 1.8 h (n = 3), while in female rats it was 42.3 ± 2.9 h (n = 3). HBenβCD increased the solubility and enhanced the dissolution rate of letrozole. Complexation of letrozole with HBenβCD improved oral absorption in male rats and maximized absorption in female rats. Regardless of gender, the presence of HBenβCD in the formulation increased the in-vivo rate of absorption. When administered in a capsule formulation with letrozole, HBenβCD resulted in a higher Cmax (61% in male rats, 42% in female), shorter T max values (8.4 to 6.3 h in male, 16.4h to 5.4 h in female) and increased absolute oral bioavailability (46 ± 2 vs 38 ± 3 in male, 101 ± 3 vs 95 ± 2 in female). Thus, solubility limits both rate and extent of letrozole absorption in male rats, but limits only the rate of absorption in female rats.

Purpose: Little is known about the mechanisms that regulate cell cycle withdrawal of the retinal pigment epithelium (RPE) during development, or about the mechanisms maintaining epithelial cell quiescence in adult retinas. The present study examines the potential role of the negative cell cycle regulator p27Kip1 in controlling RPE proliferation, using mice with targeted ablation of the p27Kip1 gene. Methods: Ocular tissues were obtained from wild-type and p27Kip1-null mice at several postnatal ages. Following aldehyde fixation, eyes were processed intact for JB-4 histology and electron microscopy. Alternatively, tissues were removed by manual or enzymatic dissection in order to obtain flat-mounts of the RPE attached to either the choroid-sclera or neural retina, respectively. Epithelial flat-mounts were either left unlabeled, in which case melanin pigment provided internal contrast, or labeled with Alexa Fluor 488-phalloidin and propidium iodide to visualize cell boundaries and nuclei, respectively. Results: Morphometric analysis using transverse plastic sections revealed a 96% increase in nuclear density and a 12% increase in thickness (apical to basal) for mutant vs. normal epithelia at postnatal day 35 (P35). These changes were not restricted to central or peripheral regions, and were uncorrelated with focal areas of dysplasia seen in the mutant neural retina. When similar tissues were viewed as flat-mounts, an observed 100% increase in nuclear density was accompanied by only a 46% enhancement in cellular density. This resulted in a larger proportion of multinucleated cells in the nullizygous RPE as compared with the wild-type epithelium (91 versus 47%). Such a pattern was achieved relatively early in development since, at P7 when the increase in RPE nuclear density was essentially complete, cellular density was augmented by only 39%. In addition to these proliferative changes, individual epithelial cells sometimes exhibited structural abnormalities, including an altered cortical actin cytoskeleton and displacement of nuclei from their normal central position. Surprisingly, while the RPE cells of null animals were similar ultrastructurally to those of the wild-type, interdigitation of their microvillous processes with outer segments was incomplete. Quantitative analysis revealed that such areas of detachment characterize, on average, 42% of the nullizygous retina, and that there is little correlation between detachment and neural retina dysplasia from one eye to another. Together with parallel evidence demonstrating a substantial decline in the apparent adhesiveness of mutant retinas relative to the normal tissue, the data is strongly indicative of an altered epithelium-photoreceptor interaction following gene ablation. Conclusions: The absence of a functional p27Kip1 gene results in enhanced RPE nuclear division, without a commensurate increase in cell division. Although the mutant epithelium as a whole appears structurally normal, individual cells exhibit cytoskeletal changes and their interaction with the neural retina is compromised.

In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells.

The aminoglycoside antibiotic hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal-subunit formation. Pulse-chase labeling experiments were performed, which verified lower rates of ribosomal-subunit synthesis in drug-treated cells. Hygromycin B exhibited a concentration-dependent inhibitory effect on viable-cell numbers, growth rate, protein synthesis, and 30S and 50S subunit formation. Unlike other aminoglycosides, hygromycin B was a more effective inhibitor of translation than of ribosomal-subunit formation in E. coli. Examination of total RNA from treated cells showed an increase in RNA corresponding to a precursor to the 16S rRNA, while mature 16S rRNA decreased. Northern hybridization to rRNA in cells treated with hygromycin B showed that RNase II- and RNase III-deficient strains of E. coli accumulated 16S rRNA fragments upon treatment with the drug. The results indicate that hygromycin B targets protein synthesis and 30S ribosomal-subunit assembly.

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection off mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcγ receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.