Importance of oxygen tension in human ovarian tissue in vitro culture.

STUDY QUESTION: Is there any difference between 20% and 5% oxygen (O2) tension in vitro culture (IVC) on the viability and quality of human follicles contained in cultured ovarian cortex? SUMMARY ANSWER: An O2 tension of 5% yields higher follicle viability and quality than does 20% O2 tension after 6 days of IVC. WHAT IS KNOWN ALREADY: The primordial follicle (PMF) pool resides within the ovarian cortex, where the in vivo O2 tension ranges between 2% and 8%. Some studies suggest that lowering O2 tension to physiological levels may improve in vitro follicle quality rates. STUDY DESIGN, SIZE, DURATION: This prospective experimental study included frozen-thawed ovarian cortex from six adult patients (mean age: 28.5 years; age range: 26-31 years) who were undergoing laparoscopic surgery for non-ovarian diseases. Ovarian cortical fragments were cultured for 6 days at (i) 20% O2 with 5% CO2 and (ii) 5% O2 with 5% CO2. Non-cultured fragments served as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cortical fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; Ki67 staining to evaluate PMF proliferation; cleaved caspase-3 immunostaining to identify follicle apoptosis; 8-hydroxy-2-deoxyguanosine and gamma-H2AX (γH2AX) immunolabeling to detect oxidative stress damage and DNA double-strand breaks (DSBs) in oocytes and granulosa cells (GCs); and ß-galactosidase staining to assess follicle senescence. Droplet digital PCR was also performed to further explore the gene expression of superoxide dismutase 2 (SOD2) and glutathione peroxidase 4 (GPX4) from the antioxidant defense system and cyclin-dependent kinase inhibitors (p21 and p16) as tissue senescence-related genes. MAIN RESULTS AND THE ROLE OF CHANCE: Apoptosis (P = 0.002) and follicle senescence (P < 0.001) rates were significantly lower in the 5% O2 group than in the 20% O2 group. Moreover, GCs in follicles in the 20% O2 group exhibited significantly (P < 0.001) higher oxidative stress damage rates than those in the 5% O2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (P = 0.001) in the 20% O2 group than in the 5% O2 group. SOD2 expression was significantly greater in the 5% O2 group compared to the 20% O2 group (P = 0.04) and the non-cultured group (P = 0.002). Expression of p21 was significantly increased in both the 20% O2 (P = 0.03) and 5% O2 (P = 0.008) groups compared to the non-cultured group. Moreover, the 20% O2 group showed significantly greater p16 expression (P = 0.04) than the non-cultured group, while no significant variation was observed between the 5% O2 and no culture groups. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study focuses on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue. The impact of O2 tension in further steps, such as secondary follicle isolation and maturation, was not investigated here. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that 5% O2 tension culture is a promising step toward potentially solving the problem of poor follicle viability after IVC. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0064.22, CDR J.0063.20 and grant 5/4/150/5 awarded to M.M.D.). The authors have nothing to disclose.

Characteristics of antimicrobial stewardship programmes in rural East Texas hospitals.

INTRODUCTION: Antimicrobial stewardship programmes (ASPs) serve as the primary method to prevent and manage the development of antimicrobial resistance. Rural settings may lack the recommended personnel and resources needed to provide antimicrobial stewardship services. METHODS: An electronic survey was distributed via e-mail to pharmacy directors or antimicrobial stewardship programme directors of licensed hospitals within Public Health Region 4/5N of East Texas. RESULTS: Sixty percent of ASPs were established <12 months prior to the survey administration. All ASPs had pharmacist involvement, with only one (5%) having formal infectious diseases (ID) training through postgraduate education. Ninety percent of ASPs had a physician champion, with five (27.8%) physicians having formal ID training. Most institutions lacked one or more recommended antimicrobial stewardship practices. When compared with ASPs established for <12 months, ASPs existing for at least 12 months were more likely to have protocols to change antimicrobials from intravenous to enteral forms (100% vs 50%, P = 0.042), provide education to patients and families on appropriate antimicrobial use (87.5% vs 33.3%, P = 0.028), and track antimicrobial purchasing costs (87.5% vs 33.3%, P = 0.028). CONCLUSIONS: Institutions in rural settings require additional resources, personnel, and time to implement ASPs and perform various antimicrobial stewardship practices.

Small RNAs Play Big Roles: MicroRNAs in Diabetic Wound Healing.

Skin and corneal wounds in diabetics are a major healthcare burden. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate the expression of protein-coding genes. Studies have identified microRNAs involved in all phases of wound healing. The dysregulation of microRNAs can contribute to impaired or delayed skin and corneal wound healing in diabetics. Here, we present a comprehensive review of the literature involving microRNAs in diabetic skin and corneal wound healing as well as those serving as potential biomarkers for diabetic wound healing.

TGF-ß1-induced transcription factor networks in Langerhans cell development and maintenance.

Langerhans cells (LC) represent a specialized subset of evolutionarily conserved dendritic cells (DC) that populate stratified epithelial tissues, which are essential for the induction of skin and mucosal immunity and tolerance, including allergy. Transforming growth factor-ß1 (TGF-ß1) has been confirmed to be a predominant factor involved in LC development. Despite great advances in the understanding of LC ontogeny and diverse replenishment patterns, the underlying molecular mechanisms remain elusive. This review focuses on the recent discoveries in TGF-ß1-mediated LC development and maintenance, with special attention to the involved transcription factors and related regulators.

Genome-wide transcriptional analysis of differentially expressed genes in flagellin-pretreated mouse corneal epithelial cells in response to Pseudomonas aeruginosa: involvement of S100A8/A9.

We previously showed that pre-exposure of the cornea to Toll-like receptor 5 ligand flagellin induces profound mucosal innate protection against infections by modifying gene expression. Taking advantage of easily procurable epithelial cell population, this study is the first report to use genome-wide cDNA microarray approach to document genes associated with flagellin-induced protection against Pseudomonas aeruginosa in corneal epithelial cells (CECs). Infection altered the expression of 675 genes (497 up and 178 down), while flagellin pretreatment followed by infection resulted in a great increase in 890 gene upregulated and 37 genes downregulated. Comparing these two groups showed 209 differentially expressed genes (157 up, 52 down). Notably, among 114 genes categorized as defense related, S100A8/A9 are the two most highly induced genes by flagellin, and their expression in the corneal was confirmed by realtime PCR and immunohistochemistry. Neutralization of S100A8 and, to a less extent, A9, resulted in significantly increased bacterial burden and severe keratitis. Collectively, our study identifies many differentially expressed genes by flagellin in CECs in response to Pseudomonas. These novel gene expression signatures provide new insights and clues into the nature of protective mechanisms established by flagellin and new therapeutic targets for reducing inflammation and for controlling microbial infection.

Safrole induces apoptosis in human oral cancer HSC-3 cells.

Phytochemicals have been used as potential chemopreventive or chemotherapeutic agents. However, there are data suggesting a mutagenic effect of some phytochemicals. We hypothesized that safrole would have anticancer effects on human oral squamous cell carcinoma HSC-3 cells. Safrole decreased the percentage of viable HSC-3 cells via induction of apoptosis by an increased level of cytosolic Ca(2+) and a reduction in the mitochondrial membrane potential (ΔΨ(m)). Changes in the membrane potential were associated with changes in the Bax, release of cytochrome c from mitochondria, and activation of downstream caspases-9 and -3, resulting in apoptotic cell death. In vivo studies also showed that safrole reduced the size and volume of an HSC-3 solid tumor on a xenograft athymic nu/nu mouse model. Western blotting and flow cytometric analysis studies confirmed that safrole-mediated apoptotic cell death of HSC-3 cells is regulated by cytosolic Ca(2+) and by mitochondria- and Fas-dependent pathways.

A role for MAP kinase in regulating ectodomain shedding of APLP2 in corneal epithelial cells.

We previously reported an increased secretion of amyloid precursor-like protein 2 (APLP2) in the healing corneal epithelium. The present study sought to investigate signal transduction pathways involved in APLP2 shedding in vitro. APLP2 was constitutively shed and released into culture medium in SV40-immortalized human corneal epithelial cells as assessed by Western blotting, flow cytometry, and indirect immunofluorescence. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC-epsilon-specific, N-myristoylated peptide inhibitor. Epidermal growth factor (EGF) also induced APLP2 accumulation in culture medium. Basal APLP2 shedding as well as that induced by PMA and EGF was blocked by a mitogen-activated protein kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-epsilon may be involved in the induction of APLP2 shedding in corneal epithelial cells.

Corneal organ culture model for assessing epithelial responses to surfactants.

The main goal of the present study was to investigate the response of cultured bovine corneas to the application of irritant substances and its potential use for predicting ocular irritancy in humans. We hypothesized that chemicals causing eye irritation may induce disruption of epithelial tight junctions and trigger cell stress responses modulated via transcription factors such as AP-1 and NF-kappaB. A simple air-lifted corneal organ culture system was used as an ex vivo model for ocular irritancy test. The effects of two surfactants, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAK), on corneal epithelial permeability and DNA-binding activity of AP-1 and NF-kappaB were studied in cultured bovine corneas. Both SDS and BAK induced tight junction disruption and increased permeability of corneal epithelium assessed using surface biotinylation in a concentration- and time-dependent manner. An increase in DNA-binding activity measured using electrophoretic mobility shift assay was observed when cultured corneas were treated with surfactants at concentrations causing minimal to mild ocular irritation, indicating epithelial cell stress response. Furthermore, exposure of cultured corneas to SDS or BAK at concentrations causing severe ocular irritancy resulted in a decrease in DNA-binding activity of these transcription factors in epithelial cells. These results indicate that the combination of corneal organ culture and measurements of corneal epithelial permeability and DNA-binding activity of stress-response transcription factors following chemical exposure has the potential to be used as a mechanistically based alternative to in vivo animal testing.

Corneal epithelial tight junctions and their response to lipopolysaccharide challenge.

PURPOSE: To investigate the expression and cellular distribution of putative tight junction (TJ) proteins occludin, ZO-1, ZO-2, and claudin-1 in rat corneal epithelium and alterations of TJs in cultured human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. METHODS: Immunohistochemistry was used to determine tissue distribution of occludin, ZO-1, ZO-2, and claudin-1 in the rat cornea. Reverse transcription-polymerase chain reaction was used to reveal the expression of mRNAs for claudins in simian virus (SV)40-immortalized human corneal epithelial (THCE) cells. To assess epithelial response to LPS challenge, THCE cells were cultured on the upper chamber of Transwell filters (Costar, Cambridge, MA), transepithelial electrical resistance (TER) was measured using a voltohmmeter. Immunocytochemistry and immunoblotting were used to assess alteration in the levels and localization of TJ-associated proteins occludin, ZO-1, and ZO-2 in LPS-treated THCE cells. RESULTS: Occludin, ZO-1, and ZO-2 were found at the cell borders of the superficial layer, whereas claudin-1 was localized mainly in the basal and wing cell layers of rat corneal epithelium. In addition to claudin-1, the transcripts for several other isotypes of claudins-2, -3, -7, -9, -14, and -15 were identified in THCE cells. Treatment of cultured THCE cells with LPS caused a dose- and time-dependent increase in monolayer permeability as assessed by TER measurements. The maximal decrease of TER was observed at approximately 6 to 9 hours after LPS challenge. The TER was then recovered gradually and returned to baseline after 24 hours. Examination of specific proteins associated with TJs by immunoblot analysis and immunomicroscopy revealed changes in the expression levels and localization of some of these proteins after their exposure to LPS. Specifically, LPS challenge resulted in a decrease in the levels of ZO-1 and ZO-2 compared with untreated cells. Reduction of the ZO-2 level was associated with the disappearance of ZO-2 staining from cell borders in 6-hour LPS-treated cells. CONCLUSIONS: Occludin, ZO-1, and ZO-2, but not claudin-1, are components of corneal epithelial TJs. LPS induces breakdown of the epithelial barrier through disruption of TJs, and ZO-1 and ZO-2 are targets for the induction.

Differential expression of MT1-MMP (MMP-14) and collagenase III (MMP-13) genes in normal and wounded rat corneas.

PURPOSE: Several members of the matrix metalloproteinase (MMP) group have been identified in the rat cornea during corneal wound healing. The aim of the present study was to identify additional members of the MMP gene family in the rat cornea and localize the expression of membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14) and collagenase III (MMP-13) in normal and wounded corneas. METHODS: Adult rats underwent laser keratectomy on the right eye. Unwounded left eyes were normal controls. Corneas were collected and processed at different times post-wounding. Reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing were used to discover the MMP genes expressed in the corneas. In situ hybridization was performed to localize the mRNA expression of MMP-14 and MMP-13. RESULTS: MMP-13 mRNA was detected in epithelial cells of wounded corneas, but not in normal controls; MMP-14 was found in both normal and wounded corneas. MMP-14 mRNA was expressed predominantly in the stromal keratocytes and rarely in the basal epithelial cells in normal and wounded corneas. MMP-13 mRNA was localized exclusively to basal cells of the epithelium at the wounded area from 6 hours to 3 days after wounding. CONCLUSIONS: MMP-14 and MMP-13 expression in rat corneas parallels that of gelatinases A and B, respectively. MMP-13 may play an important role in the gelatinase B-associated proteolytic cascade that allows rapid turnover of the extracellular matrix (ECM) components during corneal wound healing. MMP-14 may contribute to removing abnormal ECM components through activation of gelatinase A in rat corneas.

A novel epithelial wound-related gene is abundantly expressed in developing rat cornea and skin.

PURPOSE: We previously used mRNA differential display and identified a novel gene that is up regulated in the healing corneal epithelium. To understand the potential in vivo role of this gene, termed T4a, we cloned the full-length T4a cDNA and investigated its temporal and spatial transcription expression in healing rat corneas, as well as in developing cornea and skin. METHODS: The displayed T4a cDNA was used to identify clones from a rat cDNA library derived from healing corneal epithelia. The cDNA clones were sequenced and the sequence was analyzed with the Blast program. In situ hybridization was performed using digoxigenin-labeled riboprobes and cryostat sections from healing and developing cornea as well as skin. RESULTS: The T4a cDNA had 2538 bp with an open reading frame of 2178 bp, consistent with a conceptual translation product of 725 amino acid residues, a calculated molecular mass of 83.1 kD and theoretical pI of 6.93. Although T4a exhibited no sequence homology with known genes in the GenBank, it matched a large number of Expressed Sequence Tags (ESTs) from human, mouse and rat tissue cDNA libraries; more than half of the murine T4a ESTs were from embryonic DNA libraries. Sequence analysis revealed numerous phosphorylation and myristoylation sites in the deduced amino acid sequence of T4a. In the wounded rat cornea, intensive T4a mRNA staining was observed in the epithelium at all stages of re-epithelialization indicating that the expression of T4a is wound-related. In 7 day old mice, an abundant level of T4a transcripts was found in the epidermis and hair follicles, as well as in the corneal epithelial layer. High levels of T4a mRNA staining persisted in the developing postnatal corneal epithelia. In contrast, weak mRNA staining was only detected in the basal layer of the adult epidermis and corneal epithelium. CONCLUSION: These results indicate that T4a expression correlates with re-epithelialization of the cornea and maturation of the cornea and skin, suggesting a role for this gene in epithelial development, differentiation, and wound healing.

Identification of androgen receptor protein and 5alpha-reductase mRNA in human ocular tissues.

BACKGROUND/AIMS: Androgens have been reported to influence the structural organisation, functional activity, and/or pathological features of many ocular tissues. In addition, these hormones have been proposed as a topical therapy for such conditions as dry eye syndromes, corneal wound healing, and high intraocular pressure. To advance our understanding of androgen action in the eye, the purpose of the present study was twofold: firstly, to determine whether tissues of the anterior and posterior segments contain androgen receptor protein, which might make them susceptible to hormone effects following topical application; and, secondly, to examine whether these tissues contain the mRNA for types 1 and/or 2 5alpha-reductase, an enzyme that converts testosterone to the very potent metabolite, dihydrotestosterone. METHODS: Human ocular tissues and cells were obtained and processed for histochemical and molecular biological procedures. Androgen receptor protein was identified by utilising specific immunoperoxidase techniques. The analysis of type 1 and type 2 5alpha-reductase mRNAs was performed by the use of RT-PCR, agarose gel electrophoresis, and DNA sequence analysis. All immunohistochemical evaluations and PCR amplifications included positive and negative controls. RESULTS: These findings show that androgen receptor protein exists in the human lacrimal gland, meibomian gland, cornea, bulbar and forniceal conjunctivae, lens epithelial cells, and retinal pigment epithelial cells. In addition, our results demonstrate that the mRNAs for types 1 and 2 5alpha-reductase occur in the human lacrimal gland, meibomian gland, bulbar conjunctiva, cornea, and RPE cells. CONCLUSION: These combined results indicate that multiple ocular tissues may be target sites for androgen action.

Amyloid precursor-like protein 2 promotes cell migration toward fibronectin and collagen IV.

Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins.

Upregulation of xanthine oxidase by lipopolysaccharide, interleukin-1, and hypoxia. Role in acute lung injury.

LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1beta (100 microg/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1beta or hypoxia independently produced a 2-fold elevation (p < 0. 05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1beta and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1beta alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1beta, or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1beta, and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.

Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation.

Reactive oxygen species (ROS) play an important role in the pathogenesis of ischemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to anoxia-reoxygenation (A-R). To determine potential sources of intracellular ROS formation in EC in response to A-R, a fluorometric assay based on the oxidation of 2',7'-dichlorofluorescin was used. Intracellular ROS production declined 40% during 6 h of anoxia (P < 0.05). After A-R, the rates of intracellular ROS formation increased to 148 +/- 9% (P < 0.001) that of normoxic EC (100 +/- 3%). In EC exposed to A-R, allopurinol and NG-methyl-L-arginine (L-NMMA), inhibitors of xanthine oxidase (XO) and nitric oxide synthase (NOS), respectively, reduced intracellular ROS formation by 25 +/- 1% (P < 0.001) and 36 +/- 4% (P < 0.01). Furthermore, at low doses (i.e., 20 microM), deferoxamine and diethylenetriaminepentaacetic acid (DTPA) significantly inhibited intracellular ROS formation. However, at 100 microM, only deferoxamine caused further reduction in DCF fluorescence. In summary, EC respond to A-R by generating increased amounts of XO- and NOS-derived intracellular ROS. The inhibition, to a similar extent, caused by allopurinol and L-NMMA, as well as the effect of deferoxamine and DTPA suggest that the ROS detected is peroxynitrite. Based on these findings and previous work, we conclude that EC generate ROS in response to A-R from at least two different sources: a plasma membrane-bound NADPH oxidase-like enzyme that releases H2O2 extracellularly and XO, which generates intracellular O2-, which in turn may react with nitric oxide to form peroxynitrite.

Intrinsic cervical spinal cord deformation on MRI: "the distorted 'H' sign".

Extrinsic and intrinsic pathologic processes involving the spinal cord can affect its gross morphologic appearance. Contour-related abnormalities of the spinal cord can be determined by both noninvasive and invasive imaging techniques. Detailing internal dysmorphism of the spinal cord is more difficult to determine because the internal architecture of the cord is not usually visualized. Now magnetic resonance (MR) imaging can readily demonstrate the central "H" configuration of the normal spinal gray matter on axial T2* gradient-recall echo pulse sequences; thus, it should also be capable of demonstrating distortions of it. We initially reviewed 55 abnormal cervical spine 1.5-T MR imaging studies. Of 37 large lesions, 31 deformed the "H" whereas 18 small lesions did not. To compare potential differences in visualization of the "H" by MR scanners of different field strengths (1.5-0.5 T), a total of 125 additional patients were reviewed at different State University of New York (SUNY) sites. Visualization of the "H" varied from 51.4% at 1.5 T to 18.4% at 0.5 T. As resolution of the spinal cord increases on MR imaging, it becomes possible to more accurately map the altered cord "interior," which may have a detectable clinical (neurologic) counterpart.

Regulation of intracellular xanthine oxidase by endothelial-derived nitric oxide.

We have previously shown that nitric oxide (NO) donors, such as nitrosoglutathione, inhibit endothelial cell (EC) xanthine dehydrogenase (XD)/xanthine oxidase (XO) activity. The purpose of this study was to assess whether endothelial-derived NO plays any role in the regulation of intracellular XD/XO. We exposed rat pulmonary microvascular EC to L-arginine (precursor of NO) or inhibitors of nitric oxide synthase (NOS), i.e., NG-nitro-L-arginine methyl esther (L-NAME) and NG-nitro-L-arginine, in conditions of normoxia, hypoxia, and hypoxia followed by reoxygenation. Hypoxia alone caused a 1.9- and a 6.6-fold increase in XO and a 5-fold increase in XO + XD activities after 24 and 48 h of exposure, respectively. The combination of hypoxia and L-NAME (300 microM) treatment amounted at 48 h to a 10- and 7.5-fold increase in XO and XO + XD activities, respectively, compared with normoxic untreated cells. L-NAME also prevented the decline in XD/XO activity that occurred in untreated EC after hypoxia-reoxygenation. On the other hand, treatment with L-arginine caused a dose-dependent decrease in XD/XO activity in hypoxic EC compared with cells provided with L-arginine-free medium. In separate experiments, we assessed the role of L-arginine supplementation on the in vivo regulation of lung XD/XO by exposing male adult Sprague-Dawley rats for a period of 5 days to a hypoxic hypobaric atmosphere (0.5 atm). Exposure to hypoxia produced a significant increase in lung tissue XO activity and an increase in the ratio of XO to XD. L-Arginine supplementation in the drinking water prevented the increase in lung XO and the XO-to-XD ratio in hypoxic rats and caused a significant decrease in XO and XD in rats exposed to normoxia. In conclusion, this study suggests that endogenous NO has a significant role in the regulation of XD/XO both in vitro and in vivo. By inhibiting XD/XO activity, NO may have a modulating effect in conditions of hypoxia and hypoxia-reoxygenation, where this enzyme is thought to be important.

Determination of xanthine dehydrogenase mRNA by a reverse transcription-coupled competitive quantitative polymerase chain reaction assay: regulation in rat endothelial cells by hypoxia and hyperoxia.

The enzyme system xanthine dehydrogenase (XD):xanthine oxidase, which generates the superoxide anion as a by-product of action on endogenous substrates, is believed to play a role in mediating pathophysiological changes through its contribution to total superoxide production. To aid with analysis of factors that regulate XD, we have developed a reverse transcription (RT)-coupled competitive quantitative polymerase chain reaction (PCR) assay which enables XD mRNA to be determined from small amounts of cultured cells where constitutive XD levels are low. A homologous insertion mutant of wild-type XD cDNA was prepared and used as an internal standard to normalize intersample PCR efficiency differences. XD mRNA levels determined by RT-PCR also were normalized to tubulin mRNA to compensate for RT differences and loading effects among samples. We report that XD mRNA levels, determined by RT-PCR, were increased twofold in hypoxic (3% oxygen) rat pulmonary microvascular endothelial cells relative to normoxic controls (20% oxygen). Conversely, XD mRNA was decreased threefold within 24 h under hyperoxic (95% oxygen) conditions. These data support the hypothesis that XD is regulated by oxygen tension in the pulmonary vasculature.

Quantification of polymerase chain reaction products: enzyme immunoassay based systems for digoxigenin- and biotin-labelled products that quantify either total or specific amplicons.

Enzyme immunoassays were developed for quantification of polymerase chain reaction (PCR) products, referred to as amplicons. Amplicons were dual labelled simultaneously by enzymatic incorporation of digoxigenin and biotin during PCR. For total amplicon quantification, Microfluor B polystyrene wells, compatible with chemiluminescent detection, were coated with streptavidin. Dual labelled amplicons were bound, treated with anti-digoxigenin antibody conjugated to alkaline phosphatase to complete the two-site sandwich immunoassay configuration, and detected by the chemiluminescence generated upon hydrolysis of a phosphate substituted dioxetane substrate, AMPPD. For specific amplicon quantification, the Microfluor B wells were coated with an unlabelled DNA probe complementary to the labelled amplicon target. Subsequent steps were performed as described above. This assay detects 2 pg of specifically amplified DNA. Chemiluminescent detection provides a linear range of four orders of magnitude for amplicon quantification. The non-radioactive labels are safe and stable. PCR as described here obviates the need for labelled primers and constitutes the initial report of concurrent dual non-radioactive labelling of DNA by a DNA polymerase.

Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen.

Oxygen (O2) may regulate pulmonary vascular resistance through changes in endothelial nitric oxide (NO) production. To determine whether constitutive NO synthase (cNOS) is regulated by O2, we assessed cNOS expression and activity in bovine pulmonary artery endothelial cells exposed to different concentrations of O2. In a time-dependent manner, changes in O2 concentration from 95 to 3% produced a progressive decrease in cNOS mRNA and protein levels resulting in 4.8- and 4.3-fold reductions after 24h, respectively. This correlated with changes in cNOS activity as determined by nitrite measurements. Compared with 20% O2, cNOS activity was increased 1.5-fold in 95% O2 and decreased 1.9-fold in 3% O2. A decrease in O2 concentration from 94 to 3% shortened cNOS mRNA half-life from 46 to 24 h and caused a 20-fold repression of cNOS gene transcription. Treatment with cycloheximide produced a threefold increase in cNOS mRNA at all O2 concentrations, suggesting that cNOS mRNA expression is negatively regulated under basal condition. We conclude that O2 upregulates cNOS expression through transcriptional and post-transcriptional mechanisms. A decrease in cNOS activity in the presence of low O2 levels, therefore, may contribute to hypoxia-induced vasoconstriction in the pulmonary circulation.