Uptake and intracellular activity of moxifloxacin in human neutrophils and tissue-cultured epithelial cells

BACKGROUND AND PURPOSE: In light of previously reported concerns regarding carotid endarterectomy (CEA) use in our city, our goal was to determine the influence of a prospective audit and educational campaign on the performance of CEA with respect to surgical appropriateness and complication frequency. METHODS: Results of our previous audit of 291 CEAs, along with CEA practice guidelines and notification of prospective surveillance, were supplied to surgeons performing CEA in our city. After this, 184 consecutive patients undergoing CEA from September 1996 to August 1997 were followed prospectively. On the basis of blinded standardized remeasurements of angiographic carotid stenoses, CEA was classified as appropriate for patients with symptomatic carotid stenoses >/=70%, uncertain for those with symptomatic stenoses <70% or asymptomatic stenoses >/=60%, and inappropriate for patients with asymptomatic carotid stenoses <60% or preoperative neurological or medical instability. RESULTS: Forty percent of patients were asymptomatic. Compared with our prior audit, the rate of appropriate CEAs improved from 33% previously to 49% of cases in the present study (P=0.0005), uncertain indications did not change significantly (49% versus 47%; P=0.61), and inappropriate indications dropped from 18% to 4% (P=0. 00002). Perioperative stroke or death occurred in 6.4% of symptomatic patients but developed in only 2.7% of asymptomatic patients, which was improved from the 5.1% rate previously found. CONCLUSIONS: In our city, the use of a surgical audit identified areas of concern regarding CEA, and subsequent education and ongoing surveillance significantly improved the use and performance of this procedure.     

Interaction of fluorescein with the dicarboxylate carrier in rat kidney cortex mitochondria

The interaction of the organic anion, fluorescein (FL), with mitochondria in renal proximal tubule cells was investigated. Confocal microscopy was used to demonstrate FL accumulation in mitochondria of intact cells. Phenylsuccinate inhibited the mitochondrial accumulation of the FL analog, carboxyfluorescein (CF) indicating that the dicarboxylate carrier may be involved in the intracellular compartmentation of organic anions. To characterize the interaction, radio-tracer uptake and respiration studies with renal mitochondria were carried out using succinate as a substrate. Respiration measurements in freshly isolated kidney cortex mitochondria revealed that FL inhibited ADP-stimulated and uncoupled respiratory rate, indicating that the organic anion inhibited the availability of succinate as a reducing agent. A similar effect on mitochondrial respiration was found for PAH and phenylsuccinate. FL inhibited 14C-succinate uptake concentration-dependently, and Dixon analysis revealed that the nature of interaction between FL and succinate was competitive, Ki values of 0.5 +/- 0.2 and 1.1 +/- 0.8 mM were calculated for respiration experiments and tracer uptake studies, respectively. The data demonstrate that FL competitively interacts with a mitochondrial dicarboxylate transporter.     

Efflux of intracellular versus plasma membrane cholesterol in HepG2 cells: different availability and regulation by apolipoprotein A-I

We have compared the efflux of cholesterol from different cellular pools of human hepatoma cells HepG2 using intact cells or isolated membrane fractions. To label different pools, cells were incubated with either unesterified [14C]cholesterol that had been incorporated into high density lipoproteins ([14C]FC-HDL), low density lipoproteins ([14C]FC-LDL), or phosphatidylcholine liposomes ([14C]FC-PC), or with [14C]acetate. Cell fractionation revealed that labeling of cells with [14C]FC-PC resulted in the incorporation of [14C]cholesterol almost exclusively into the plasma membrane (PM), while incubation with [14C]FC-HDL resulted in the majority of [14C]cholesterol incorporation into the PM, but with a smaller component associated with lysosomes. Labeling with [14C]FC-LDL or [14C]acetate led to an accumulation of [14C]cholesterol predominantly in lysosomes or the endoplasmic reticulum (ER), respectively. When the kinetics of [14C]cholesterol efflux was analyzed after pulse-labeling of different cellular pools, half-times of cholesterol efflux from lysosomes and ER were significantly longer than that from PM. In another set of experiments, when both labeling and efflux times varied, efflux of [14C]cholesterol from the PM to human serum after 1.5 h pulse and chase incubations was double that from lysosomes and 8-fold that from ER. Extension of the incubation times from 1.5 to 3 h diminished the difference in cholesterol efflux from different membranes. Further incubation to 6 h almost abolished the different responses. Cell-free preparations of membranes, obtained from cells labeled with [14C]cholesterol, showed no differences in cholesterol efflux. No differences in the distribution of [14C]cholesterol released into serum among lipoprotein subfractions was observed. Pretreatment of the serum with Fab fragments of polyclonal rabbit anti-human apolipoprotein A-I antibodies reduced its ability to promote efflux of cholesterol from the ER by 77%, but had no effect on cholesterol efflux from the PM. Fab fragments of non-immune IgG had no effect on the efflux of both ER and PM cholesterol. We conclude that the availability of cellular cholesterol for efflux from HepG2 cells is strongly influenced by its subcellular location, and is regulated by apolipoprotein A-I.     

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.     

Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.     

Modulation of intracellular glutathione and cysteine metabolism in bovine oviduct epithelial cells cultured In vitro

The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/ microg protein for Cys and from 5.3 to 6.4 pmol/ microg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 microM. Both compounds depleted GSH in a dose-dependent manner, and 500 microM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 microM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 microM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.     

Kinetic analysis of transcytosis of epidermal growth factor in Madin-Darby canine kidney epithelial cells

OBJECTIVE: Treatment outcome in sexually abused preschool children was evaluated 6 and 12 months after treatment. METHOD: Forty-three sexually abused preschool children and their parents were evaluated 6 and 12 months after completion of either Cognitive-Behavioral Therapy for Sexually Abused Preschoolers (CBT-SAP) or nondirective supportive therapy (NST). Parents completed the Child Behavior Checklist, Child Sexual Behavior inventory, and Weekly Behavior Report to measure a variety of symptoms in their children. RESULTS: Repeated-measures analyses indicated that there were significant group by time interactions on several outcome measures from the beginning of the study to the end of the 12-month follow-up period, with the CBT-SAP group exhibiting significantly more improvement over time than the NST group. Clinical findings also indicated the superior effectiveness of CBT-SAP over NST in reducing sexually inappropriate behavior. CONCLUSIONS: Findings support the superior efficacy of CBT-SAP over NST in maintaining symptom reduction in the year after treatment completion. The importance of using cognitive-behavioral interventions for sexually inappropriate behaviors and including nonoffending parents in the treatment of sexually abused preschool children is discussed.     

In vitro stimulation of renal tubular p-aminohippurate transport by dexamethasone in kidney tissue of immature and adult rats

STUDY OBJECTIVES: To evaluate the automated determination of onset and offset times and amplitudes of all the PQRST waves from simultaneously recorded surface electrocardiogram (SECG) and unipolar esophageal ECG (EsECG). The occurrence of ST segment deviation is also examined. DESIGN: Prospective, observational study. SETTING: University hospital. PATIENTS: 30 patients undergoing coronary artery bypass graft (CABG) surgery. INTERVENTIONS: SECG and two-lead unipolar EsECG were recorded after induction of anesthesia and before cardiopulmonary bypass (CPB). MEASUREMENTS AND MAIN RESULTS: The amplitudes of the P and T waves and the ST segment deviation were measured. EsECG had more noise than SECG. Slight movement of the esophageal electrodes occasionally caused substantial changes in the wave amplitudes and ST segment deviation in the unipolar EsECG. The maximum P wave amplitude in EsECG was, on average, 97% greater than the maximum P wave amplitude in SECG, ST segment deviation in EsECG was observed in the absence of ST segment deviation in SECG and vice versa. CONCLUSIONS: The recognition and measurement of all the PQRST waves can be improved and automated by simultaneous use of EsECG and SECG. The P wave amplitude is greater in EsECG than in SECG, which may faciliate the identification of supraventricular versus ventricular arrhythmias. ST segment deviation in the unipolar EsECG may not be suitable for the routine detection of ischemia.     

IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability

The nitric oxide (NO) signaling system, consisting of NO synthases, soluble guanylyl cyclase, and cGMP, plays a prominent role in salt handling and regulation of blood pressure. Soluble guanylyl cyclases are heme-containing heterodimers (alpha/beta). The alpha1/beta1 isoform has greater NO sensitivity than the alpha1/beta2. It has recently been shown that expression of the beta subunits is altered in the kidney of the Dahl salt-sensitive rat, ie, the beta1 subunit is decreased and the beta2 subunit increased. However, whether soluble guanylyl cyclase is linked to salt sensitivity is not known. In the present study, we investigated linkage of guanylyl cyclase genes to blood pressure. Alpha1 and beta1 gene loci for soluble guanylyl cyclase were mapped to rat chromosome 2, and the beta2 gene locus was mapped to rat chromosome 5 using fluorescent in situ metaphase hybridization. By use of a rat radiation hybrid panel, the gene loci were then further mapped with respect to known quantitative trait locus markers of salt-sensitive hypertension in the Dahl rat on chromosomes 2 and 5. Genes for alpha1 and beta1 were closely linked by two-point analysis to Na+,K+-ATPase alpha1 isoform (LOD of 15.1 and 14.0, respectively) and calmodulin-dependent protein kinase II-delta loci (LOD of 14.3 and 12.9, respectively), which have been previously shown to flank a quantitative trait locus for blood pressure in the Dahl rat. The alpha1 and beta1 genes were closely linked (LOD of 11.3; theta, 0.4). The beta2 gene locus was closely linked to the endothelin-2 (ET-2) locus (LOD of 13.0), which has been shown to cosegregate with blood pressure. We conclude that soluble guanylyl cyclase subunit loci, ie, alpha1, beta1, and beta2, are good candidates for genes controlling salt-sensitive hypertension in the Dahl rat.     

Accumulation of ochratoxin A in rat kidney in vivo and in cultivated renal epithelial cells in vitro

Biological applications of triplex forming oligonucleotides will require the development of oligomers with high avidity and specificity. We examined the binding enhancement resulting from intercalator conjugation to both parallel design (polythymidine T15) and antiparallel design (polypurine AG15, for binding a 15 base pair polypurine-polypyrimidine sequence in the IL-2R alpha gene enhancer) oligomers under various ionic strength and temperature conditions. Oligonucleotides were conjugated through a urea link to 6,9 diamino-3-methoxy acridine (to give T15C and AG15C). Intercalator conjugation dramatically enhanced the specific triplex binding avidity (Kd = 5 nM for AG15C and 275 nM for T15C at 25 degrees C, compared to 2 microM for AG15 and > 50 microM for T15 at 25 degrees C), without detectable binding to an inappropriate target sequence. Surprisingly, triplex formation with AG15C occurred at lower Mg2+ concentrations than with T15C. AG15 and AG15C showed rapid Mg2+ dependent self association, but not T15C or T15. T15C triplex formation occurred rapidly (completion in less than 4 min), while AG15C bound to its target sequence more slowly over 20-24 h. Thus, binding constants in the low nanomolar range are now achievable with intercalator conjugated polypurine antiparallel binding oligonucleotides, a prerequisite for biological applications of such agents.     

Cholinergic inhibition of Na-K-ATPase via activation of protein kinase C in Madin-Darby canine kidney cells

Recently, it was reported that muscarinic-type cholinergic receptors coupled to the phosphoinositide messenger system are present in the rabbit inner medullary collecting duct and Madin-Darby canine kidney (MDCK) cells. The receptor density in MDCK cells is 50 times more than that in inner medullary collecting duct cells. To examine if muscarinic receptor activation influences Na-K-ATPase, the effects of a cholinergic agonist, carbachol, on Na-K-ATPase activity in MDCK cells were measured. Carbachol inhibited Na-K-ATPase activity in a time- and concentration-dependent manner. A maximum of approximately 80% of the enzyme activity was inhibited in 160 min with an EC50 of 5 microM carbachol. The inhibition of Na-K-ATPase activity was reversible; up to 80% of the enzyme activity was recovered within 4 h after carbachol was removed. The inhibitory effect of carbachol was blocked by a muscarinic antagonist atropine and by inhibitors of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine HCl, and N-(2-(methylamino)ethyl)-5-isoquinoline sulfonamide HCl. Direct activators of PKC, phorbol 12-myristate 13-acetate, N(n-heptyl)-5-chloro-1-naphthalene sulfonamide, and phosphatidyl serine, also inhibited Na-K-ATPase activity in MDCK cells, and their effect was also blocked by PKC inhibitors. These results indicate that cholinergic agonists inhibit Na-K-ATPase activity in MDCK cells by the activation of PKC. It is concluded that the inhibition of Na-K-ATPase by PKC may, in part, be responsible for the natriuretic action of cholinergic agonists, which have been shown to stimulate phosphoinositide hydrolysis in renal collecting duct cells.     

Calreticulin: an intracellular Ca++-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells

Calreticulin was identified in a screen for androgen-response genes in the rat ventral prostate. Northern blot and Western blot analyses in the rat model showed that both calreticulin messenger RNA and protein are down-regulated by castration and up-regulated by androgen replacement in the prostate. Northern blot analysis showed that calreticulin expression level in the prostate is much higher than that in seminal vesicles, heart, brain, muscle, kidney, and liver. The regulation of calreticulin expression by androgen is only observed in the prostate and seminal vesicles, two male secondary sex organs. The induction of calreticulin by androgen in prostate organ culture partially resists protein synthesis inhibition, suggesting that calreticulin is a direct androgen-response gene. In situ hybridization and immunohistochemistry studies showed that calreticulin is an intracellular protein in prostatic epithelial cells. Because calreticulin is a major intracellular Ca++-binding protein with 1 high-affinity and 25 low-affinity Ca binding sites, our observations suggest that calreticulin is a promising candidate that mediates androgen regulation of intracellular Ca++ levels and/or signals in prostatic epithelial cells. The expression of calreticulin is also regulated by androgen in the mouse and human prostate, suggesting that androgen regulation and function of calreticulin in the prostate are conserved evolutionarily.     

Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca2+ and cell proliferation in primary human mammary epithelial cells

A method was developed to determine in eggs 2 components [4,6-dimethyl-2-hydroxypyrimidine and 1,3-bis(4-nitrophenyl)urea] of the anticoccidial drug nicarbazin, used to treat poultry. Samples were extracted with acetonitrile, and the extracts were washed with hexane and evaporated to dryness before analysis by liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization. By switching from positive to negative ion monitoring and using gradient elution, both components were measured within one run. The limit of quantitation of the assay was 10 ng/g for each component. The results of a preliminary feeding trial in which chickens were fed contamination levels of the drug are also reported.     

Membrane type 1-matrix metalloproteinase is involved in the formation of hepatocyte growth factor/scatter factor-induced branching tubules in madin-darby canine kidney epithelial cells

The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell cycle and a potential tumor suppressor gene. Because we had previously demonstrated that loss of p27 protein is associated with aggressive behavior in colorectal adenocarcinomas, we used immunohistochemistry and in situ hybridization to evaluate the potential role of alterations in p27 expression in primary and metastatic colorectal adenocarcinomas. Parallel immunostaining was performed for Ki-67 and p53. We evaluated 13 cases of metachronous and 23 cases of synchronous primary and metastatic colorectal tumor pairs. In the synchronous subgroup (Stage IV tumors), 57% of the primary tumor and metastases pairs did not express p27 protein and the remainder were low expressors. In the metachronous subgroup, 54% of the primary tumors were low expressors and the remainder high expressors of p27 protein. There was a significant reduction in the expression of p27 in the metachronous metastases (mean positive cells: 14.5%) when compared to the corresponding primary tumors (mean positive cells: 41.8%), P = 0.0023. All the primary and metastatic tumors in the metachronous subgroup showed high levels of p27 mRNA expression. There was no association between loss of p27 and either Ki-67 count or p53 expression. Because p27 is known to be up-regulated when epithelial cells are grown in suspension, the down-regulation of p27 in circulating tumor cells may confer the ability to grow in an environment of altered extracellular matrix or intercellular adhesion properties, two situations which may facilitate metastases.     

Increased functional load on mouse kidney proximal tubule epithelial cells causes changes in nucleolar 3-D architecture

Ultrastructural 3-D analysis of nucleolar architecture and Ag-NOR protein distribution in mouse kidney-cortex proximal-tubule epithelium has been performed. A principal scheme of structural changes of the nucleolus and organization of its components during the intensification of pre-rRNA synthesis (dynamic model of a nucleolus) based on computer spatial modelling has been advanced. According to the nucleolar composition, three groups of cells, which differ from each other by rRNA synthesis, are defined in normal kidney. Most nephron proximal-section cells (about 52%) are characterized by lower activity of RNA synthesis. Such kind of cells are defined as group I (nucleolar diameter 0.7-1.5 microm) and always contain resting, ring-shaped or close to ring-shaped dense nucleoli, which have 2 or 3 fibrillar centers. Nucleoli of group II cells (about 37%, nucleolar diameter 1.5-2.5 microm) have a higher level of activity, contain 4-7 fibrillar centers, and their structural organization is close to reticulated forms due to the first indications of vacuolar network (identified as prereticulated nucleoli). The most active cells of group III (about 11%, nucleolar diameter 2.5-3.5 microm) include cells with typical reticulated nucleoli with a well expressed vacuolar network and numerous fibrillar centers (18-22). Increased functional load of the epithelium caused by unilateral nephrectomy and diuretic (4-chlor-H [2-furylmethyl] 5-sulphamyl-antranic acid) injection changed the proportion of the different cell groups: group I decreased (about 25%), whereas groups II and III increased (about 8% and 17%, respectively). The increase of nucleolar activity first causes a deformation of the individual fibrillar centers as well as complication and growth of their surface. Further, a progressive fragmentation of the fibrillar centers and the growth of their total volume is observed. The complication and growth of the total volume of Ag-positive zones is another indication of the nucleolar activation. The vacuolar system develops by a gradual fusion of small isolated cavities into a united vacuolar network. Nucleoli with 2-7 fibrillar centers are considered to be intermediate forms reflecting successive stages of its activation or inactivation: from the resting ring-shaped nucleolus via transient stages of increasing functional activity to the active reticulated nucleoli and vice versa. The observed differences in the nucleolar ultrastructure are regarded as evidence of the functional heterogeneity of cell populations within one functional segment of nephron.