Modeling the response of a biofilm to silver-based antimicrobial.

Biofilms are found within the lungs of patients with chronic pulmonary infections, in particular patients with cystic fibrosis, and are the major cause of morbidity and mortality for these patients. The work presented here is part of a large interdisciplinary effort to develop an effective drug delivery system and treatment strategy to kill biofilms growing in the lung. The treatment strategy exploits silver-based antimicrobials, in particular, silver carbene complexes (SCC). This manuscript presents a mathematical model describing the growth of a biofilm and predicts the response of a biofilm to several basic treatment strategies. The continuum model is composed of a set of reaction-diffusion equations for the transport of soluble components (nutrient and antimicrobial), coupled to a set of reaction-advection equations for the particulate components (living, inert, and persister bacteria, extracellular polymeric substance, and void). We explore the efficacy of delivering SCC both in an aqueous solution and in biodegradable polymer nanoparticles. Minimum bactericidal concentration (MBC) levels of antimicrobial in both free and nanoparticle-encapsulated forms are estimated. Antimicrobial treatment demonstrates a biphasic killing phenomenon, where the active bacterial population is killed quickly followed by a slower killing rate, which indicates the presence of a persister population. Finally, our results suggest that a biofilm with a ready supply of nutrient throughout its depth has fewer persister bacteria and hence may be easier to treat than one with less nutrient.

Nanoparticle deposition onto biofilms.

We develop a mathematical model of nanoparticles depositing onto and penetrating into a biofilm grown in a parallel-plate flow cell. We carry out deposition experiments in a flow cell to support the modeling. The modeling and the experiments are motivated by the potential use of polymer nanoparticles as part of a treatment strategy for killing biofilms infecting the deep passages in the lungs. In the experiments and model, a fluid carrying polymer nanoparticles is injected into a parallel-plate flow cell in which a biofilm has grown over the bottom plate. The model consists of a system of transport equations describing the deposition and diffusion of nanoparticles. Standard asymptotic techniques that exploit the aspect ratio of the flow cell are applied to reduce the model to two coupled partial differential equations. We perform numerical simulations using the reduced model. We compare the experimental observations with the simulation results to estimate the nanoparticle sticking coefficient and the diffusion coefficient of the nanoparticles in the biofilm. The distributions of nanoparticles through the thickness of the biofilm are consistent with diffusive transport, and uniform distributions through the thickness are achieved in about four hours. Nanoparticle deposition does not appear to be strongly influenced by the flow rate in the cell for the low flow rates considered.

Female Wistar-Kyoto and SHR/y rats have the same genotype but different patterns of expression of renin and angiotensinogen genes.

OBJECTIVES: To evaluate whether renin and angiotensinogen gene expression in females from two strains of rats that share the same autosomes and X chromosomes differs. Female SHR/y rats have the parental Wistar-Kyoto rat autosomes and X chromosomes and have no chromosomes of spontaneously hypertensive rat origin; thus they are genetically equivalent to female Wistar-Kyoto rats. DESIGN AND METHODS: Because these genes are regulated by steroid hormones, we investigated the effects of removal of estrogen (ovariectomy) and addition of androgen (testosterone implants) on three groups of female SHR/y rats and the parental rat strain Wistar-Kyoto rat with groups of intact (control) rats, rats subjected to ovariectomy at age 3 weeks, and rats subjected to ovariectomy with a testosterone implant at age 3 weeks. RESULTS: The combination of removing estrogen early in development and supplementing the ovariectomized females with testosterone revealed strain differences in response of blood pressure. Renin and angiotensinogen messenger RNA levels appear to be regulated coordinately within each strain, although actual levels of messenger RNA differ between the strains. CONCLUSIONS: Similar patterns of responses of renin and angiotensinogen genes to ovariectomy and ovariectomy plus testosterone suggest that regulation of the genes is likely to be similar or coordinate. Differences in regulation of renin-angiotensin system genes between strains may result from epigenetic mechanisms such as genome imprinting of these genes or of another gene that functions as a common regulator of renin and angiotensinogen.

In vitro evaluation of electrostatic endothelial cell transplantation onto 4 mm interior diameter expanded polytetrafluoroethylene grafts.

PURPOSE: To perform an in vitro evaluation of electrostatic endothelial cell transplantation of human umbilical vein endothelial cells (HUVEC) onto segments of 4 mm internal diameter expanded polytetrafluoroethylene (ePTFE) vascular prostheses. METHODS: This evaluation consisted of exposing vascular graft segments that had been subjected to either electrostatic or gravitation transplantation with HUVEC to a physiologic shear stress (15 dynes/cm2) under steady flow conditions within a flow loop system. Biochemical assays were performed on freshly transplanted grafts by means of radioimmunoassay for prostacyclin and thromboxane A2. RESULTS: There was a 30% loss of HUVEC after 30 minutes of shear stress exposure from the grafts subjected to gravitational transplantation with no additional significant (alpha = 0.05) loss after 120 minutes. Grafts subjected to electrostatic transplantation had no significant (alpha = 0.05) loss of HUVEC during exposure to physiologic shear stress. Furthermore, after 120 minutes of shear-stress exposure, the grafts subjected to electrostatic transplantation (78,420 +/- 6274 HUVEC/cm2) retained 2.3 times more HUVEC than the counterparts subjected to gravitational transplantation (34,427 +/- 4637 HUVEC/cm2). The biochemical assay results indicated no significant (alpha = 0.05) production of prostacyclin or thromboxane A2 regardless of the method of cell transplantation. CONCLUSIONS: (1) The electrostatic transplantation technique was superior to the gravitational transplantation technique in terms of cellular retention when the ePTFE grafts were exposed to physiologic shear stress. (2) Production of prostacyclin and thromboxane A2 did not differ between transplanted HUVEC subjected to gravitational or electrostatic procedures.

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA.

We have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen has not previously been available, Expression noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because greater than 99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing us to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

The transcriptional response of the human chorionic gonadotropin beta-subunit gene to cAMP is cycloheximide sensitive and is mediated by cis-acting sequences different from that found in the alpha-subunit gene.

Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP and phorbol esters interact synergistically to regulate expression of the chorionic gonadotropin genes.

Previous studies have shown that activators of the protein kinase A pathway increase transcription of the genes encoding the alpha- and beta-subunits of human chorionic gonadotropin (hCG) in choriocarcinoma cell lines. Here, we show that treatment of choriocarcinoma cells with activators of protein kinase C, such as phorbol myristate acetate (PMA) and dioctanoylglycerol, increases accumulation of the mRNAs for both subunits of hCG by 3-4-fold. In contrast, a phorbol ester which fails to activate protein kinase C, phorbol 12 beta,13 alpha-didecanoate, has no effect on hCG mRNA levels. To test the possibility that these two major intracellular signaling pathways interact, we treated choriocarcinoma cells with PMA, forskolin, or PMA and forskolin together. Treatment with either agent led to a 2-3-fold increase in hCG mRNA levels, whereas treatment with both agents resulted in a 9-fold increase. This synergistic response also occurred when choriocarcinoma cells were treated with PMA and 8-Br-cAMP. Furthermore, PMA did not increase intracellular cAMP levels, suggesting that these two pathways interact subsequent to cAMP generation. PMA also increased transcription of the hCG alpha- and beta-genes by 2-3-fold. Whereas transcription of the alpha subunit gene increases synergistically after treatment with both PMA and forskolin, transcription of the hCG beta-gene was limited to the increase caused by either agent alone. This latter result suggests that regulation of hCG beta mRNA accumulation is more complex than that of alpha-subunit mRNA and probably involves both transcriptional and post-transcriptional components.

17 Beta-estradiol and progesterone inhibit transcription of the genes encoding the subunits of ovine follicle-stimulating hormone.

FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein subunits, alpha and beta. It is known that 17 beta-estradiol (E2) and progesterone (P4) can decrease the secretion and synthesis of FSH in ovine pituitary cultures. Data presented here indicate that E2 and P4 decrease the steady state levels of FSH beta mRNA concomitantly with FSH secretion in ovine pituitary cultures. By 24 h, E2 decreased the steady state levels of FSH beta mRNA and FSH secretion by 68% +/- 5%. P4 also decreased both concomitantly, but by 58% +/- 7% after 24 h. E2 and P4 also decrease steady state levels of alpha mRNA, but at a lower rate. Finally, it is shown that E2 and P4 decrease transcription of the FSH beta by greater than 85% in 2 h; alpha mRNA transcription is decreased by 70% in 12 h. These effects are not altered even when cycloheximide is present to block protein synthesis by 95%. These data further define the mechanisms whereby E2 and P4 inhibit ovine FSH secretion/synthesis directly at the pituitary level. They also provide the first example of negative transcriptional regulation by P4 and the second of two examples now established for E2.

Phenotypes of HeLa S3 variant cell lines resistant to growth inhibition by sodium butyrate.

HeLa cell variants capable of multiplying in the presence of sodium butyrate were used to study the relationship of cell cycle position to human chorionic gonadotropin (hCG) production and regulation of the genes encoding hCG alpha- and beta-subunits. The butyrate-resistant variants exhibit several different stable phenotypes. In wild-type HeLa cells, butyrate arrests cell division and modulates synthesis of alpha- and beta-subunits of glycoprotein hormones by coordinately regulating steady-state levels of their respective mRNAs. Because the variant cell lines replicate, in addition to producing hCG subunits in the presence of butyrate, cell cycle arrest does not seem to be a requirement for expression of glycoprotein hormone genes. Studies of histone modification suggest that neither hyperacetylation of histones H3 and H4 nor dephosphorylation of histones H1 and H2A mediates inhibition of cell replication. In the variants, alpha-subunit and hCG beta levels are independently regulated, as a consequence of independent regulation of alpha- and beta-hCG mRNA levels. Long-term effects of butyrate include derepression of some genes (hCG beta in the variant AO) and repression of others (hCG alpha in variant AO). Moreover, hormone production correlates with the steady-state levels of mRNA for each of the subunits, suggesting that regulation occurs before translation. These findings indicate that the butyrate-resistant variant cell lines are valuable for studies of the molecular mechanisms involved in regulation of expression of ectopic hormones.

Cyclic AMP regulates transcription of the genes encoding human chorionic gonadotropin with different kinetics.

Choriocarcinoma cell lines characteristically synthesize and secrete the alpha- and beta-subunits of human chorionic gonadotropin (hCG), as well as the intact heterodimer. Treatment of one such cell line, BeWo, with 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP) causes at least a 10-fold increase in the concentration of the mRNA encoding each subunit. Changes in mRNA concentrations are associated with similar changes in transcription rates of both the CG alpha and CG beta genes, although the kinetics of their transcriptional responses are different. Transcription of the alpha-subunit gene increases rapidly and becomes maximal within 1 hr after addition of 8-Br-cAMP. By contrast, transcription of the CG beta gene increases slowly and progressively for at least 8 hr after treatment with 8-Br-cAMP. The slow transcriptional response of the CG beta gene(s) appears to be unique compared to that of other cAMP-responsive genes, and suggests that the cyclic nucleotide may regulate transcription of the CG genes by different mechanisms.

Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element.

cAMP regulates transcription of the gene encoding the alpha-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the alpha-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the alpha-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the alpha-subunit gene.

Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study.

Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-1) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg++-adenosine triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1. The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in wnterphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells. The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubles. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.