Effect of carbohydrates on the ability of bull sperm to bind to bovine oviduct epithelial cells.

In the present study, we investigated the effect of various carbohydrates on the ability of bovine spermatozoa to bind to the bovine oviduct epithelial cells (OECs). We also examined the fertilization competence and motility of spermatozoa that bind to OECs in the presence of carbohydrates. Frozen-thawed spermatozoa were incubated with OECs, with and without various carbohydrates. The sperms were then divided into two fractions: OEC-binding sperms (B-sperm) and non-OEC binding sperms (NB-sperm). The fertilization rate, ability to bind the zona pellucida, and membrane integrity of the spermatozoa as determined using a hypo-osmotic-swelling test (HOST) were lower in NB-sperm than in the unseparated spermatozoa (control). The motility of the B-sperm was maintained for a longer time than that of the control spermatozoa. The addition of N-acetyl-d-glucosamine (GlcNAc, 5 mm) to the sperm-OEC mixture increased the number of B-sperm. D-mannose (5 mm) and D-fucose (5 mm) had no effect on the number of B-sperm. The motility of B-sperm, which bound to OECs in the presence of GlcNAc, however, was not maintained. When either OECs or the spermatozoa were treated with GlcNAc prior to sperm-OEC co-incubation, only sperm-side treatment enhanced sperm-OEC binding, but B-sperm motility was not maintained. The motility of spermatozoa incubated with GlcNAc was lower than that of controls. These results indicate that GlcNAc enhances sperm binding to OECs, probably via sperm surface modification, but does not promote increased sperm survival.

Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos.

The duration of sperm-oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm-oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (>5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1-3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%; P<0.05), and the highest rate of normal fertilization was observed for 5h culture (73.4%; P<0.05). The sex ratio was significantly (P<0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP; P<0.05) and decreased the proportion of HOST positive sperm (65.8-48.6%; P<0.05; experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5-10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1h (treatment 5) or 5h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively; P<0.05), and the 1h co-incubation significantly skewed (P<0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%; P<0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9; 1.0 mg/ml treatment 10) significantly (P<0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus-sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes.

Inclusion of the transcervical approach in video-assisted thoracoscopic extended thymectomy (VATET) for myasthenia gravis: a prospective trial.

BACKGROUND: Because evidence-based data regarding the quality of video-assisted thoracoscopic thymectomy for the treatment of myasthenia gravis are lacking, a prospective trial comparing three different operative approaches was conducted to evaluate their efficacy. METHODS: This prospective study enrolled 20 consecutive patients with nonthymomatous myasthenia gravis. A series of three approaches for bilateral video-assisted thoracoscopic extended thymectomy (VATET) using the anterior chest wall-lifting method (original), the original method with a flexed-neck position (modified), and the original method with a transcervical approach (final) were prospectively performed in each patient for quantitative and pathologic evaluation of the residual thymus after each approach. RESULTS: Complete VATET required 242 +/- 48 min, with the transcervical procedure requiring 23 +/- 12 min. After the modified method, the residual thymus in the cervical region was 1.5 cm in size and weighed 0.8 g (0.8% of the entire thymus), as compared with a size of 2.2 cm and a weight of 1.3 g (3.2%) after the original method. Each value is the result of comparison with the final method. Histopathologic studies showed residual tissue in the germinal center as well as Hassall's corpuscles in more than 70% of cases. CONCLUSION: The findings show that VATET without the transcervical approach could be an immunologically incomplete treatment for myasthenia gravis. Therefore, the transcervical approach should be included in VATET procedures to ensure radicality.

Safer video-assisted thoracoscopic thymectomy after location of thymic veins with multidetector computed tomography.

BACKGROUND: Video-assisted thoracoscopic (VATS) thymectomy has been applied as a surgical option for autoimmune myasthenia gravis. Prior identification and fine division of the thymic veins are critical to the prevention of unexpected severe bleeding that may require conversion to open surgery. Until recently, such bleeding could be avoided only by meticulous dissection of thymic fat tissue away from the left brachiocephalic vein (LBV). With recent advances in computed tomography (CT), multidetector-row computed tomography (MDCT) can readily be obtained and provides three-dimensional (3D) images. This study explored its value for preoperative identification of the thymic veins draining into the LBV, and thus for prevention of injury to these veins during endoscopic thymectomy. METHODS: Five patients with myasthenia gravis, thymoma, or both underwent enhanced MDCT preoperatively. The thymic veins draining into the LBV were visualized using both horizontal and sagittal/coronal CT images. Then 3D images were reconstructed to enable operators to simulate endoscopic views. During each VATS extended thymectomy, the numbers and branching patterns of the thymic veins were compared with the preoperative MDCT images. RESULTS: The thymic veins draining into the LBV were clearly identified with MDCT in all five patients examined. Reconstructed 3D images clearly located their courses in the thymic/fat tissue and their entry routes into the LBV, thus simulating the actual intraoperative endoscopic views. All tributaries divided during surgery were identified preoperatively with MDCT. CONCLUSIONS: Location of thymic veins with MDCT can provide precise preoperative information about thymic venous anatomy. This easy and less invasive examination has the potential to make VATS thymectomy easier and safer.

Regulation of nuclear import by light-induced activation of caged nuclear localization signal in living cells.

A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine-128 residue in SV40 T-antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78+/-0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm(2)). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally.

Clinical and functional significance of WHO classification on human thymic epithelial neoplasms: a study of 146 consecutive tumors.

We examined the clinical and functional significance of histologic classification of thymic epithelial neoplasms proposed by the World Health Organization (WHO), based on an analysis of 146 consecutive tumors derived from 141 patients and 47 normal thymuses derived from children ranging in age from 1 to 9 years. Invasive tumors were seen in 12.5%, 38.6%, 40.0%, 69.4%, 80.0%, and 100% of type A, AB, B1, B2, B3, and C primary tumors, respectively. All of six recurrent or metastatic lesions were type B2 tumors. Myasthenia gravis was associated in 0%, 6.8%, 40.0%, 55.6%, 10.0%, and 0% in patients with type A, AB, B1, B2, B3, and C tumors, respectively. The average number (x10(6)) of tumor-associated CD4+CD8+ cells present in 1 g of tumor tissue was 1.5, 391.1, 1041.7, 333.9, 24.5, and 0.2 in type A, AB, B1, B2, B3, and C, respectively, and it was 1168.2 in the normal thymuses. Thus, type B1 tumor retained the function to induce CD4+CD8+ double-positive cells at a level comparable to that of the normal thymic cortical epithelial cells, followed by type AB and type B2 tumors. Type A and B3 tumors had this function at a barely detectable level, and type C tumor was nonfunctional. WHO histologic classification was shown to reflect the clinical features and the T-cell-inducing function of thymic epithelial tumors.

Maleimidoethyl 3-(tri-n-butylstannyl)hippurate: a useful radioiodination reagent for protein radiopharmaceuticals to enhance target selective radioactivity localization.

In pursuit of radiolabeled monoclonal antibodies (mAbs) with rapid urinary excretion of radioactivity from nontarget tissues, radioiodinated mAbs releasing a m-iodohippuric acid from the mAbs in nontarget tissues were designed. A novel reagent, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH), was synthesized by reacting N-(hydroxyethyl)maleimide with N-Boc-glycine before coupling with N-succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE). MIH possessed a maleimide group for mAb conjugation and a butylstannyl moiety for high-yield and site-specific radioiodination, and the two functional groups were linked via an ester bond to release m-iodohippuric acid. To investigate the fate of radiolabels after lysosomal proteolysis, hepatic parenchymal cells were used as a model nontarget tissue and 131I-labeled MIH was conjugated with galactosyl-neoglycoalbumin (NGA). Further conjugation of [131I]MIH with a mAb against osteogenic sarcoma (OST7) after reduction of its disulfide bonds was followed up. In murine biodistribution studies, [131I]MIH-NGA exhibited rapid accumulation in the liver followed by radioactivity elimination from the liver at a rate that was identical to and faster than those of 131I-labeled NGA via direct iodination ([131I]NGA) and [131I]ATE-labeled NGA, respectively. While [131I]NGA indicated high radioactivity levels in the murine neck, stomach, and blood, such increases in the radioactivity count were not detectable by the administration of either [131I]MIH-NGA or [131I]ATE-NGA. At 6 h postinjection of [131I]MIH-NGA, 80% of the injected radioactivity was recovered in the urine. Analyses of urine samples indicated that m-iodohippuric acid was the sole radiolabeled metabolite. In biodistribution studies using [131I]-MIH-OST7 and [131I]ATE-OST7, while both 131I-labeled OST7s registered almost identical radioactivity levels in the blood up to 6 h postinjection, the former demonstrated a lower radioactivity level than [131I]ATE-OST7 in nontarget tissues throughout the experiment. Such chemical and biological characteristics of MIH would enable high target/nontarget ratios in diagnostic and therapeutic nuclear medicine using mAbs and other polypeptides.

Nitric oxide production by bronchoalveolar cells during allograft rejection in the rat.

BACKGROUND: We reported the increased nitric oxide (NO) level in exhaled air of rat lung transplant recipients during acute rejection (AR). The aim of this study was to determine the site and level of NO production in the rejected graft. METHODS: Rat lung transplantation was performed in isografts and allografts. RESULTS: In isografts, no AR and no significant increase in NO production was identified. In allografts, grades I-II of AR was seen on postoperative day (POD) 3 and grade III on POD 5. NO produced by BAL cells increased on both POD 3 (11.8+/-2.0 parts per billion [ppb]) and POD 5 (115.3+/-66.9 ppb). There was a highly significant correlation between the level of NO and the severity of AR (p=0.862, P<0.005). BAL cells from allografts expressed iNOS mRNA. Among them, macrophages, lymphocytes and neutrophils were immunostained for iNOS. CONCLUSION: NO produced by BAL cells was detected in the early stages of rejection. Therefore, it may serve as a sensitive indicator of AR in lung transplantation.

Reduced dorsal hippocampal glutamate release significantly correlates with the spatial memory deficits produced by benzodiazepines and ethanol.

Memory deficits frequently occur after taking benzodiazepines and ethanol. We studied in vivo hippocampal presynaptic glutamate transmission in conjunction with memory deficits induced by benzodiazepines and ethanol in rats as an animal model of amnesia. These drugs potently impaired spatial memory formation as evaluated by the Morris water maze task, the rank order among tested treatments being the combination of triazolam (20 micrograms/kg) with ethanol (2 g/kg) > or = triazolam (100 micrograms/kg) > ethanol (2 g/kg) > or = triazolam (20 micrograms/kg) > rilmazafone (20 micrograms/kg). On the other hand, these drug treatments also reduced glutamate release in the dorsal hippocampus but not in the cerebellum measured by microdialysis: the combined administration of triazolam with ethanol potently inhibited glutamate release to 60% of basal output in the dorsal hippocampus. These decreases in hippocampal glutamate transmission closely correlated with the extent of impairment of spatial memory performance (r = 0.990). Thus, the present results strongly indicated that presynaptic dysfunction in dorsal hippocampal glutamatergic neurons would be critical for spatial memory deficits induced by benzodiazepines and ethanol.

The questionnaire to parents of children with the Down syndrome: how to inform the parents and psychological responses to counseling.

This study determined the experience of 137 sets of parents when they were informed that their child had Down syndrome and how they would have preferred this matter to have been handled. The survey revealed that the majority of parents would have preferred being told as soon as possible, with both of them present, and that they had suspected something wrong at the birth of the child. This information prompted us to analyze critically the parental experiences and to formulate a positive approach, with sensitive, supportive, and progressive counseling.

Tetraploidy in a 15-month-old girl.

We present a 15-month-old girl with tetraploidy and compare the manifestations with those of 3 previously reported liveborn infants with the same type of polyploidy. Common anomalies noted included micro-turricephaly, a prominent but narrow forehead, microphalmia or anophthalmia, limb anomaly, sacral meningomyelocele, and mental retardation.

Diagnosis of twin zygosity by hypervariable RFLP markers.

The application of variable number of tandem repeat (VNTR) markers to the determination of twin zygosity was investigated. In the first case, which was performed with the use of six VNTR markers, the probability of monozygosity, calculated from Essen-Möller's formula II, was 0.99972. In the other three cases in which four VNTR markers were analyzed, the probabilities were 0.98251-0.99557. These results suggest that VNTR markers are useful for determination of twin zygosity.

Scenarios for autoimmunization of T and B cells in myasthenia gravis.

We have studied responses in thymoma patients to interferon-alpha and to the acetylcholine receptor (AChR) in early-onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two-step process: (step 1) professional antigen-presenting cells and thymic epithelial cells prime AChR-specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross-react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen-antibody complexes and the recruitment of professional antigen-presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.

Cellular contraction precedes membrane depolarization in Vorticella convallaria

Application of a mechanical stimulus to the cell body of the peritrich ciliate Vorticella convallaria evoked an all-or-nothing membrane depolarization, the large pulse. This was always accompanied by an all-or-nothing cellular contraction, and simultaneous recordings of the two events revealed that the large pulse was always preceded by the cellular contraction. A smaller graded membrane depolarization (the medium pulse) was sometimes produced in response to a weaker mechanical stimulus. The medium pulse was accompanied by a small, graded, localized contraction of the cell body and was occasionally followed by a large pulse. When a large pulse occurred during a medium pulse, it reached the same peak level as that of a large pulse evoked without a preceding medium pulse. When a medium pulse occurred during a medium pulse, summation of the two pulses was observed. Sustained contraction causes V. convallaria to become rounded, and in this state a mechanical stimulus stronger than that used to evoke the large pulse evoked a graded depolarizing mechanoreceptor potential in the cell. We conclude that both the large and medium pulses are caused by an inward receptor current that is activated mechanically following contraction of the cell body. A localized contraction evokes a small mechanoreceptor current, causing a medium pulse. An all-or-nothing contraction evokes a saturated, all-or-nothing mechanoreceptor current, causing a large pulse.

Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats.

The herbicide paraquat is an environmental factor that could be involved in the etiology of Parkinson's disease. We have previously shown that paraquat penetrates through the blood-brain barrier and is taken up by neural cells. In this study, we examined the in vivo toxic mechanism of paraquat to dopamine neurons. GBR-12909, a selective dopamine transporter inhibitor, reduced paraquat uptake into the striatal tissue including dopaminergic terminals. The subchronic treatment with systemic paraquat significantly decreased brain dopamine content in the striatum and slightly in the midbrain and cortex, and was accompanied by the diminished level of its acidic metabolites in rats. When paraquat was administered through a microdialysis probe, a transitory increase in the extracellular levels of glutamate, followed by long-lasting elevations of the extracellular levels of NO(x)(-) (NO(2)(-) plus NO(3)(-)) and dopamine were detected in the striatum of freely moving rats. This dopamine overflow lasted for more than 24 h after the paraquat treatment. Dopamine overflow was inhibited by N(G)-nitro-L-arginine methyl ester, dizocilpine, 6,7-dinitroquinoxaline-2,3-dione and L-deprenyl. The toxic mechanism of paraquat involves glutamate induced activation of non-NMDA receptors, resulting in activation of NMDA receptor-channels. The influx of Ca(2+) into cells stimulates nitric oxide synthase. Released NO would diffuse to dopaminergic terminals and further induce mitochondrial dysfunction by the formation of peroxynitrite, resulting in continuous and long-lasting dopamine overflow. The constant exposure to low levels of paraquat may lead to the vulnerability of dopaminergic terminals in humans, and might potentiate neurodegeneration caused by the exposure of other substances, such as endogenous dopaminergic toxins.

Potential bioactivated neurotoxicants, N-methylated beta-carbolinium ions, are present in human brain.

Potential bioactivated neurotoxicants, 2-N-methyl-beta-carbolinium and 2,9-N,N'-dimethyl-beta-carbolinium ions, as well as N-methylation activities which form these charged species, were analyzed for the first time in the parietal association cortex and the substantia nigra of human brain using GC/MS and HPLC. The brains were taken during forensic autopsies from corpses without obvious degeneration of substantia nigra. In the cortex, 2-methyl-norharmanium ion (2-MeNH) and 2,9-dimethyl-norharmanium ion (2,9-Me2NH) were detected in almost all samples. 2-Methyl-harmanium ions (2-MeHA) and 2,9-dimethyl-harmanium ions (2,9-Me2HA) were detectable in only two samples. In substantia nigra samples pooled from 3 or 4 brains for analysis, 2-MeNH and 2,9-Me2NH levels were higher than those in the cortex, whereas 2-MeHA and 2,9-Me2HA were below detection limits. Their precursors, norharman (NH) and harman (HA), were also measured using HPLC/fluorescence detection. In both regions, NH and HA were present in almost all samples; levels of NH and HA were also significantly higher in the nigra than in the cortex. Using 9-methyl-NH and 2-MeNH as substrates, in vitro N-methylation of the 2[beta] and 9[indole] nitrogens toward beta-carbolines was measured both in the cortex and in the nigra. 2[beta]-N-Methylation activity was significantly higher than 9[indole]-N-methylation activity in both regions. Recent studies show that beta-carbolinium ions resemble the synthetic parkinsonian toxicant, MPP+, with respect to structure and neurotoxic activity. Such 'bioactivated' carbolinium ions could be endogenous causative factors in Parkinson's disease.

Carrier-mediated processes in blood--brain barrier penetration and neural uptake of paraquat.

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringer's solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

T-cell development in human thymoma.

Human thymoma is derived from thymic epithelial cells and often associated with a large number of cortical thymocytes. Since thymic epithelial cells play key roles in T-cell development in the normal thymus, we hypothesized that the neoplastic epithelial cells of thymoma may support T-cell differentiation. We attempted to reconstitute the T-cell development in vitro by using neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on a proportion of CD4-CD8- cells in thymoma. These CD34+CD4-CD8- cells also expressed both IL-7R alpha-chain and common gamma-chain. Purified CD4-CD8- cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4+CD8+ cells via CD4 single positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. The CD34+CD4-CD8- cells purified from a normal thymus also differentiated to CD4+CD8+ cells in an allogeneic co-culture with the neoplastic epithelial cells of thymoma. In addition, a pleural dissemination from thymoma contained a large amount of cortical thymocytes. These results suggest that the neoplastic epithelial cells retain the function of thymic epithelium and can support T-cell development in thymomas.

Beta adrenergic antagonist permeation across cultured rabbit corneal epithelial cells grown on permeable supports.

PURPOSE: To determine whether cultured rabbit corneal epithelial cells (RCEC), grown on permeable supports, provide a suitable in vivo model for characterizing transcellular drug permeation and metabolism. METHODS: Primary rabbit corneal epithelial cells grown in DMEM-F12 were seeded on Transwell-COL inserts coated with fibronectin. The epithelial barrier integrity was evaluated, based on measurements of 14C-mannitol and 3H-PEG900, and their transepithelial electrical resistance (TEER). Ultrastructure evaluation was based on scanning electron microscopy and transmission electron microscopy, which were performed 8 days after seeding. Measurements of beta adrenergic antagonist permeability were performed to assess transcellular permeability. RESULTS: Eight days after seeding, the TEER reached a peak of 144 omega.cm2 and the 14C-mannitol and 3H-PEG900 permeabilities were 6.8 x 10(-6) and 2.9 x 10(-6) cm/sec, respectively. Ultrastructural analysis revealed a multilayered structure with numerous microplicae and typical cytoplasmic organelles along with desmosomes. The relationship between permeation of beta-blockers and lipophilicity resembled the intact isolated cornea. CONCLUSIONS: This is the first description of cultured RCEC grown on permeable support. Many of its properties mimic those described in the intact corneal epithelium. Even though its electrical tightness is less than that of the intact cornea, the transcellular permeability to lipophilic beta-antagonists is comparable to the isolated preparation. Therefore, this model will facilitate characterization of ocular permeation mechanisms of hydrophobic drugs whose route of permeation is transcellular.

Characterization of the carrier-mediated transport of levofloxacin, a fluoroquinolone antimicrobial agent, in rabbit cornea.

The cornea presents a formidable barrier to drug penetration. The fluoroquinolone levofloxacin, which is an effective antimicrobial agent, has the potential to be used in the topical treatment of ocular disease. Thus, we sought to characterize how levofloxacin penetrates the cornea. To perform this characterization, we measured the time dependent permeation of levofloxacin across the isolated rabbit cornea using a diffusion chamber, and compared it with antipyrine fluxes. Levofloxacin permeation into the receiver epithelial-side bathing solution (pH = 6.5) from the donor endothelial-side (pH = 7.4) reached 3.00 nmolcm(-2) cornea after 2h, whereas in the opposite direction permeation was 1.89 nmolcm(-2) cornea. Based on the temperature-dependent effects on permeation, the calculated energy of activation for permeation, Ea, was 31.3 kcal mol(-1), whereas Ea for antipyrine, a marker of diffusion, was 11.0 kcalmol(-1). The transport of levofloxacin from epithelium to endothelium was concentration-dependent and had both a linear and saturable component. Evaluation of the kinetic parameters, Jmax, apparent Km and k(d) showed that they were 38.78 pmol min(-1) cm(-2), 3.83 mM and 0.0135 microL min(-1) cm(-2), respectively. These results, coupled with the fact that levofloxacin permeation reached a maximum value at pH 6.5, suggest that levofloxacin transport across the cornea is carrier mediated. However, at present, it cannot be ascertained whether such a system is localized in either the corneal epithelial or the endothelial layer.