Claudin-2-dependent Changes in Noncharged Solute Flux Are Mediated by the Extracellular Domains and Require Attachment to the PDZ-scaffold

Paracellular transport through the tight junction shows selectivity for both ionic charge and solute size. It is known that charged residues on the extracellular loops of claudins control charge selectivity. It is also known that inducible expression of claudin-2, but not claudin-4, will selectively increase the permeability for PEG molecules which are <4Å in radius, but it is not known whether permeability is controlled by the same regions of claudins which control charge selectivity. Using inducible expression of chimeras of claudin-2 and claudin-4 in monolayers of MDCK II cells we show that the extracellular loops alone are responsible for controlling the permeability for noncharged PEGs as well as for charge selectivity. Further, the cytoplasmic C-terminal PDZ-binding motif is required for wild type claudin-2 to control permeability, suggesting a requirement for attachment to the PDZ scaffold in order to form pores. These observations support a model where the loops form pores controlling permeability for both charged and noncharged solutes which are smaller than 4Å. They leave unanswered why both claudin-2 and -4 can influence electrical properties while only -2 can selectively increase permeability for small PEGs

ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin

Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3 nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities

Development of a liquid chromatography–tandem mass spectrometry assay of six antimicrobials in plasma for pharmacokinetic studies in premature infants

This method provides a simple extraction procedure, as well as a validated, sensitive, and specific liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of ampicillin, piperacillin, tazobactam, meropenem, acyclovir, and metronidazole in human plasma. The method was validated over concentration ranges specific for each compound, with a lower limit of quantification of 50–300 ng/mL and a sample volume of 50 μL. The method is accurate and precise, with within- and between-day accuracy ranging from 85–110% and 92–110%, respectively, and within- and between-day precision of 89–111% and 91–109%, respectively. Simplicity, low plasma volume, and high throughput make this method suitable for clinical pharmacokinetic studies in premature infants

CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}- nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine if differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of an oxygen into the amidine C=N to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of nitric oxide. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules

Prostatic Acid Phosphatase Is Required for the Antinociceptive Effects of Thiamine and Benfotiamine

Peer reviewe

Studies on Antiretroviral Drug Concentrations in Breast Milk: Validation of a Liquid Chromatography-Tandem Mass Spectrometric Method for the Determination of 7 Anti-Human Immunodeficiency Virus Medications

Studying the pharmacokinetics of antiretroviral drugs in breast milk has important implications for the health of both the mother and the infant, particularly in resource-poor countries. Breast milk is a highly complex biological matrix, yet it is necessary to develop and validate methods in this matrix, which simultaneously measure multiple analytes, as women may be taking any number of drug combinations to combat human immunodeficiency virus infection. Here, we report a novel extraction method coupled to high-performance liquid chromatography and tandem mass spectrometry for the accurate, precise, and specific measurement of 7 antiretroviral drugs currently prescribed to infected mothers. Using 200 µL of human breast milk, simultaneous quantification of lamivudine (3TC), stavudine (d4T), zidovudine (ZDV), nevirapine (NVP), nelfinavir (NFV), ritonavir, and lopinavir was validated over the range of 10–10,000 ng/mL. Intraday accuracy and precision for all analytes were 99.3% and 5.0 %, respectively. Interday accuracy and precision were 99.4 % and 7.8%, respectively. Cross-assay validation with UV detection was performed using clinical breast milk samples, and the results of the 2 assays were in good agreement (P = 0.0001, r = 0.97). Breast milk to plasma concentration ratios for the different antiretroviral drugs were determined as follows: 3TC = 2.96, d4T = 1.73, ZDV = 1.17, NVP = 0.82, and NFV = 0.21

The density of small tight junction pores varies among cell types and is increased by expression of claudin-2

Epithelial tight junctions contain size- and charge-selective pores that control the paracellular movement of charged and noncharged solutes. Claudins influence the charge selectivity and electrical resistance of junctions, but there is no direct evidence describing pore composition or whether pore size or density differs among cell types. To characterize paracellular pores independent of influences from charge selectivity, we profiled the ;apparent permeabilities' (P(app)) of a continuous series of noncharged polyethylene glycols (PEGs) across monolayers of five different epithelial cell lines and porcine ileum. We also characterized P(app) of high and low electrical resistance MDCK cell monolayers expressing heterologous claudins. P(app) profiling confirms that the paracellular barrier to noncharged solutes can be modeled as two distinct pathways: high-capacity small pores and a size-independent pathway allowing flux of larger solutes. All cell lines and ileum share a pore aperture of radius 4 A. Using P(app) of a PEG of radius 3.5 A to report the relative pore number provides the novel insight that pore density along the junction varies among cell types and is not necessarily related to electrical resistance. Expression of claudin-2 results in a selective increase in pore number but not size and has no effect on the permeability of PEGs that are larger than the pores; however, neither knockdown of claudin-2 nor overexpression of several other claudins altered either the number of small pores or their size. We speculate that permeability of all small solutes is proportional to pore number but that small electrolytes are subject to further selectivity by the profile of claudins expressed, explaining the dissociation between the P(app) for noncharged solutes and electrical resistance. Although claudins are likely to be components of the small pores, other factors might regulate pore number

Plasma Bile Acid Concentrations in Patients with Human Immunodeficiency Virus Infection Receiving Protease Inhibitor Therapy: Possible Implications for Hepatotoxicity

To evaluate whether patients with human immunodeficiency virus (HIV) infection who were receiving protease inhibitor therapy had altered bile acid concentrations compared with noninfected control subjects, and whether bile acid concentrations could predict the onset of hepatotoxicity caused by protease inhibitors

Pilot study of rosiglitazone as an in vivo probe of paclitaxel exposure: Short report

To evaluate the use of rosiglitazone and the erythromycin breath test (ERMBT), as probes of CYP2C8 and CYP3A4, respectively, to explain inter-individual variability in paclitaxel exposure