Biomedical subjects

V H Lee

Publications and source records attributed to V H Lee.

At least 19 recordsLinked to original sources

PubMed2002

Gene expression, cell localization, and evolution of rodent submandibular gland androgen-binding protein.

A small dimeric androgen-binding protein (Abp) secreted by mouse (Mus) submandibular salivary glands has been hypothesized to function in mate-selection. The alpha-subunit (Abpa) evolves rapidly under natural selection. However, cellular site(s) of synthesis, mode of function, and patterns of evolution of this biologically important protein are otherwise unknown. We used a radiolabeled riboprobe and in situ hybridization to mouse (Mus) Abpa mRNA to localize Abpa synthesis to submandibular gland acinar cells. We next used a quantitative fluorescent thermal cycler (real-time PCR) to determine relative expression levels (normalized with a constitutively expressed ribosomal gene [S15/rig; rat insulinoma gene]) in male, female, and sexually immature European striped field mice (Apodemus agrarius). We hypothesized that gender or age-related differences might occur in production of a salivary protein related to sexual selection, but found no significant differences within our sample. Finally, we also used reverse transcription-PCR of mRNAs isolated from submandibular salivary glands to determine the Abpa allele in the striped wood mouse and compare it to published information on the homologue in Mus musculus domesticus.

Amino Acid Sequence↗

PubMed2001

Pharmacogenomics of drug transporters: the next drug delivery challenge.

Scientifically, the third millennium begins with a major triumph--the publishing of the human genomic map, which is destined to have a momentous impact on the quality of life in our time. Disease prevention, individualized medicine, and genotyped-based medicine will soon become a reality. Pharmacogenetics, the forerunner of pharmacogenomics, began in the 1950s with a series of observations relating drug response to various genetic factors. It took almost two more decades for scientists to discover that cytochrome p450 2D6 was responsible for the metabolism of many drugs. This landmark discovery helped focus attention on how gene expression could impact the response to drugs. The stage was set for a revolution in therapeutics some 30 years later as the Human Genome Project crossed the finishing line triumphantly. A parallel development in drug delivery that may also benefit from the fruits of the Human Genome Project is the growing acceptance/awareness of drug transporters as a gateway to epithelial drug transport. This presentation addresses an area in need of attention: the possible impact of genetic polymorphism of drug transporters in pharmacokinetics and the challenge it poses in drug delivery.

Animals↗

PubMed2001

Delivery systems for penetration enhancement of peptide and protein drugs: design considerations.

This paper discusses the challenges to be met in designing delivery systems that maximize the absorption of peptide and protein drugs from the gastrointestinal and respiratory tracts. The ideal delivery system for either route of administration is one that will release its contents only at a favorable region of absorption, where the delivery system attaches by virtue of specific interaction with surface determinants unique to that region and where the delivery system travels at a rate independent of the transitory constraints inherent of the route of administration. Such a delivery system, which is as yet unavailable, will benefit not only peptide and protein drugs, but other poorly absorbed drugs.

Administration, Oral↗

PubMed2001

Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries.

A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (<1200 Da) cytosolic materials. These channels are a growing family of related proteins. This study was designed to determine the ontogeny of connexin 43 (Cx43) during early stages of follicular development in prepubertal porcine ovaries. A partial-length (412 base) cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with published porcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adult porcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth.

Age Factors↗

PubMed2001

Mucosal drug delivery.

This review focuses on epithelial drug transport mechanisms in mucosal drug delivery: the final step of a four-part process. Reference is made to the mucosae lining the oral cavity and the gastrointestinal tract, the two mucosae most often succumbing to the side effects of cytotoxic chemotherapeutic drugs. This review will be devoted to carrier-mediated transport, particularly as it relates to the intestinal dipeptide transporter PepT1. This transporter protein appears to be enriched in tumor epithelial cells, to be rather robust to the cytotoxic effects of chemotherapeutic drugs, and to lend itself to the molecular engineering of drugs that target this transporter in tumor epithelial cells. In contrast to the gastrointestinal tract, much less is known about the type and capacity of drug transport processes in the buccal epithelial cells and about how these processes may be altered in disease state (including cancer) and be manipulated pharmaceutically to optimize drug absorption.

Animals↗

PubMed2000

The cardiac sodium channel mRNA is expressed in the developing and adult rat and human brain.

Expression of the rat (RH-I/SkM2) and human (hH1/SCN5A) tetrodotoxin-resistant (TTX-R), voltage-sensitive sodium channels is thought to be specific to cardiac tissue. We detected RH-I/SkM2 mRNA in newborn rat brain using both RNase protection assay analysis and in situ hybridization and in adult rat brain using RNase protection assay analysis. This expression was observed primarily in developing limbic structures of the cerebrum and diencephalon, and in the medulla of the brain stem. Using RT-PCR analysis, we detected hH1/SCN5A mRNA in both fetal and adult human brain. Interestingly, mutations in the human cardiac sodium channel are known to lead to cardiac abnormalities, which result in arrhythmias and frequently in sudden cardiac death. If these mutant channels were also expressed in limbic regions of the brain, alterations in channel function could have drastic effects on the brain's signaling ability, possibly promoting seizure activity.

Adult↗

PubMed2000

Meeting future challenges in topical ocular drug delivery: development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drug transport studies.

The purpose of this study was to develop and characterize a functional air-interfaced primary culture of rabbit conjunctival epithelial cells grown on a permeable support for drug transport studies. Conjunctival epithelial cells from the pigmented rabbit were isolated, seeded at 1.2 x 10(6) cells cm(-2) on permeable Transwell filters, and cultured at the air interface using a modified PC-1 medium. Conjunctival epithelial cell layers showed a transepithelial resistance of 1.1+/-0.1 kOmega cm(2), a potential difference of 17.0+/-0.5 mV, and an equivalent short-circuit current (I(eq)) of 16.1+/-0.4 microA cm(-2). The I(eq) was reduced by 35% using 0.01 mM bumetanide, 66% using 0.1 mM ouabain, 46% using 2 mM barium chloride (all three in the basolateral fluid), and 63% using 0.3 mM NPAA in the apical fluid, consistent with active Cl(-)-secretion across the conjunctival epithelial barrier. Amiloride-sensitive Na(+) channels were absent. The permeability of the cell layers to polar solutes decreased with increased solute size, and the calculated equivalent pore size was about 8.0 nm. The Papp of beta-blockers varied with lipophilicity in a sigmoidal fashion. Uridine transport showed temperature sensitivity and directionality, favoring transport in the apical-to-basolateral direction. Apical L-carnosine uptake was reduced by 46% in the absence of an inwardly directed proton gradient, and lowering the temperature to 4 degrees C abolished direction-dependent L-carnosine uptake. Furthermore, uptake was inhibited by 73% using apical 10 mM glycyl sarcosine (a dipeptide transporter substrate) and by 60% using 1 mM L-valacyclovir (a dipeptide prodrug). In conclusion, a functional air-interfaced primary culture of rabbit conjunctival epithelial cell layers was established. This air-interfaced primary culture model may be useful for studying passive and active transport processes for ion and solute translocation in the mammalian conjunctival epithelial barrier in a defined experimental setting.

Adrenergic beta-Antagonists↗

PubMed2000

Hormonal regulation and cellular distribution of connexin 32.2 and connexin 32.7 RNAs in the ovary of Atlantic croaker.

The in vitro effects of human chorionic gonadotropin (hCG) on ovarian connexin (Cx) 32.2 and 32.7 RNA levels and ovarian follicle maturation were assessed, and the cellular distribution of Cx transcripts in the ovary was determined. hCG caused a concentration-dependent induction of Cx32.2 RNA, which peaked coincidentally with the appearance of morphological indices of oocyte maturational competence (OMC). Cx32.2 RNA levels declined thereafter in all treatment groups, although this decline was not accompanied by the onset of germinal vesicle breakdown (GVBD) at the lowest hCG concentration used. The levels of Cx32.7 RNA initially declined and subsequently increased to preincubation values after hCG treatment, but these changes were not dependent on hCG concentration. In a separate experiment, the decline in Cx32.7 RNA occurred in the presence or absence of hCG and was prevented by low (physiological) concentrations of estradiol-17beta (E2) or by protein kinase C (PKC) inhibitor, but was enhanced in the presence of high E2 concentrations or of PKC activator. These changes in Cx32. 7 RNA abundance were not associated with any indices of oocyte maturation. In situ hybridization of tissue sections showed the presence of Cx32.2 and Cx32.7 RNA in somatic cells of the ovarian follicle but not in oocytes. Cx32.2 RNA seemed to be present in granulosa and thecal cells, but the assay resolution was insufficient to reliably determine the distribution of Cx32.7 transcript by somatic cell type. In view of earlier findings that Cx32.2-based (but not Cx32.7-based) connexons can form functional homotypic channels, these results indicate that Cx32.2 gene expression in granulosa cells is sufficient for the formation of homologous gap junctions (GJ). Northern blot of RNA extracts from ovulated eggs, which are free of follicle cells, showed the presence of relatively low levels of both Cx RNAs. Thus, it is possible that Cx32.2 is present in oocytes and that it participates in heterologous (homotypic) GJ formation between the oocyte and the granulosa cells. In conclusion, Cx32.2 RNA levels in somatic cells of the ovarian follicle correlated positively with morphological indices of OMC acquisition, but subsequently declined during GVBD. These changes in Cx32.2 RNA may function in the regulation of GJ contacts during follicular maturation.

Animals↗

PubMed2000

Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva.

We determined net fluid secretion rate across the pigmented rabbit conjunctiva in the presence and absence of pharmacological agents known to affect active Cl- secretion and Na+ absorption. Fluid flow across a freshly excised pigmented rabbit conjunctiva mounted between two Lucite half chambers was measured by a pair of capacitance probes in an enclosed cabinet maintained at 37 degrees C and a relative humidity of 70%. Fluid transport was also measured in the presence of compounds known to affect active Cl- secretion (cAMP, UTP, and ouabain), Na+ absorption (D-glucose), or under the Cl--free condition on both sides of the tissue. Net fluid secretion rate across the pigmented rabbit conjunctiva in the serosal-to-mucosal direction at baseline was 4.3+/-0.2 microl/hr/cm2 (mean +/- s.e.m.). Net fluid secretion rate was increased approximately two-fold by mucosally applied 1 mM 8-Br cAMP (8.4+/-0.4 microl/hr/cm2) and 10 microM UTP (9.8+/-0.6 microl/hr/cm2), but was abolished by either serosally applied 0.5 mM ouabain (0.3+/-0.1 microl/hr/cm2) or under the Cl--free conditions (0.06+/-0.04 microl/hr/cm2). Mucosal addition of 20 mM D-glucose decreased net fluid secretion rate to 1.0+/-0.5 microl/hr/cm2. In conclusion, the pigmented rabbit conjunctiva appears to secrete fluid secondary to active Cl- secretion. This net fluid secretion is subject to modulation by changes in active Cl- secretion rate and in mucosal fluid composition such as glucose concentration.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2000

Membrane transporters.

Carrier-mediated drug transport is relatively unexplored in comparison with passive transcellular and paracellular drug transport. Yet, there is a host of transporter proteins that can be targeted for improving epithelial drug absorption. Generally, these are transport mechanisms for amino acids, dipeptides, monosaccharides, monocarboxylic acids, organic cations, phosphates, nucleosides, and water-soluble vitamins. Among them, the dipeptide transporter mechanism has received the most attention. Dipeptide transporters are H(+)-coupled, energy-dependent transporters that are known to play an essential role in the oral absorption of beta-lactam antibiotics, angiotensin-converting enzyme (ACE) inhibitors, renin inhibitors, and an anti-tumor drug, bestatin. Moreover, several investigators have demonstrated the utility of the dipeptide transporter as a platform for improving the oral bioavailability of drugs such as zidovudine and acyclovir through dipeptide prodrug derivatization. Thus far, at least four proton-coupled peptide transporters have been cloned. The first one cloned was PepT1 from the rabbit small intestine. The focus of this presentation will be structure-function, intracellular trafficking, and regulation of PepT1. Disease, dietary, and possible excipient influences on PepT1 function will also be discussed.

Animals↗

PubMed2000

Role of P-glycoprotein in restricting propranolol transport in cultured rabbit conjunctival epithelial cell layers.

PURPOSE: To determine the role of P-glycoprotein (P-gp) in propranolol transport in cultured rabbit conjunctival epithelial cell layers (RCEC). METHODS: The localization of P-gp in the cultured RCEC as well as in the excised conjunctiva was determined by immunofluorescence technique. The role of P-gp in transepithelial transport and uptake of propranolol in conjunctival epithelial cells cultured on Transwell filters was evaluated in the presence and absence of P-gp competing substrates, an anti-P-gp monoclonal antibody (4E3 mAb), or a metabolic inhibitor, 2,4-dinitrophenol (2,4-DNP). RESULTS: Immunofluorescence studies revealed positive staining in the apical membrane of cultured RCEC and in the apical surface of the superficial cell layers in the excised conjunctiva, but not the basolateral membrane of cultured RCEC. Transport of propranolol showed preference in the basolateral-to-apical direction. The net secretory flux was saturable with a Km of 71.5 +/- 24.0 nM and a Jmax of 1.45 +/- 0.17 pmol/cm2/hr. Cyclosporin A, progesterone, rhodamine 123, verapamil, 4E3 mAb and 2,4-DNP all increased apical 50 nM propranolol uptake by 43% to 66%. On the other hand, neither beta-blockers (atenolol, metoprolol, and alprenolol) nor organic cation transporter substrates (tetraethylammonium (TEA) and guanidine), affected apical 50 nM propranolol uptake. CONCLUSIONS: The energy-dependent efflux pump P-gp appears to be predominantly located on the apical plasma membrane of the conjunctival epithelium. It may play an important role in restricting the conjunctival absorption of some lipophilic drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Air-interface condition promotes the formation of tight corneal epithelial cell layers for drug transport studies.

PURPOSE: To identify the growth conditions that would favor the development of a functional primary culture of pigmented rabbit corneal epithelial cells on a permeable support comparable to the intact tissue in bioelectric properties. METHODS: Rabbit corneal epithelial cells were isolated and cultured on precoated fibronectin/collagen/laminin permeable filters. Cells were grown at an air-interface with supplemented DMEM/F12 medium. Immunofluorescence and electron microscopy techniques, respectively, were used to confirm cornea-specific marker and morphological features. Permeability of the cell layers to model polar compounds was evaluated using 14C-mannitol, fluorescein isothiocyanate (FITC) and fluorescein isothiocyanate-dextran of 4,000 molecular weight (FD4). RESULTS: We found that culturing the epithelial cells at an air-interface (AIC) was a critical factor in the formation of tight cell layer and that omitting fetal bovine serum and keeping the concentration of epidermal growth factor at 1 ng/ml were equally important. Phenotypically, the AIC cell layers were found to express cornea-specific 64 kD keratin. Compared with cells cultured under the liquid-covered (LCC) condition, those cultured under AIC exhibited a significantly higher peak transepithelial electrical resistance (TEER) of up to 5 kOhm x cm2, a higher potential difference (PD) of up to 26 mV, and an estimated short-circuit current (Ieq) of 5 microA/cm2 after 7-8 days of culture. These values were comparable to those in the excised cornea. Consistent with the TEER, the AIC cell layers were 4-40 times less permeable to paracellular markers than their LCC counterpart. CONCLUSIONS: The AIC model merits further characterization of drug transport mechanisms as well as drug, formulation, physiological, and pathological factors influencing corneal epithelial drug transport.

Animals↗

PubMed2000

Expression of RUSH transcription factors in developing and adult rabbit gonads.

The RUSH transcription factors 1alpha and 1beta bind to the Rabbit Uteroglobin promoter and are members of the SWI/SNF complex that facilitates transcription by remodeling chromatin (Helicase). To characterize gonadal expression of RUSH, a cRNA probe that recognizes both isoforms was used for in situ hybridization studies. We found RUSH mRNA to be abundant in Sertoli cells from embryonic, neonatal, prepubertal, and pubertal rabbit testes. In adults, RUSH mRNA was detected in tubules with preleptotene spermatocytes and mature spermatids lining the lumen. However, RUSH was undetectable in tubules that contained leptotene spermatocytes and that lacked mature spermatids. In females, RUSH was expressed in presumptive granulosa cells of embryonic and neonatal ovaries before follicle organization. Abundant RUSH mRNA was detected in granulosa and theca cells surrounding preantral follicles of prepubertal and adult ovaries. Expression of RUSH remained high in granulosa cells of antral follicles in mature ovaries but was negligible in late-stage atretic follicles and in corpora lutea. Western blot analysis confirmed the RUSH-1alpha isoform predominated in both testicular and ovarian tissues. The expression pattern of RUSH indicates transcriptional activity in Sertoli cells and during multiple stages of differentiating granulosa cells, especially those of primordial follicles, which heretofore were considered to be dormant.

Animals↗

PubMed2000

Expression of rabbit zona pellucida-1 messenger ribonucleic acid during early follicular development.

Progress in research on initiation of folliculogenesis has progressed slowly because of a lack of markers for early folliculogenesis. The rabbit zona pellucida protein (ZP1) is synthesized in follicles during early stages of folliculogenesis. In order to establish ZP1 as a marker for initiation of folliculogenesis, in situ hybridization was used to localize ZP1 mRNA in immature follicles. ZP1 mRNA was first detected in oocytes of some but not all primordial follicles. The primordial follicles expressing ZP1 mRNA were located at the cortico-medullary junction, indicating that they were newly activated follicles. ZP1 mRNA accumulated in oocytes of intermediate, primary, and secondary follicles. In contrast, ZP1 mRNA was first detectable in granulosa cells of intermediate follicles and is present in cuboidal granulosa cells of primary and early secondary follicles, but was undetectable in granulosa cells of more mature follicles. These data demonstrate that 1) ZP1 mRNA is expressed in both oocytes and granulosa cells, 2) ZP1 mRNA is initially expressed in oocytes of activated follicles, and 3) ZP1 mRNA is transiently expressed in granulosa cells during early stages of folliculogenesis. Therefore, rabbit ZP1 is a molecular marker that can be used in future studies to measure initiation of folliculogenesis.

Animals↗

PubMed2000

Rates of protein transport across rat alveolar epithelial cell monolayers.

The transport of model proteins, ranging from 12,300 to 150,000 Da, across tight rat alveolar epithelial cell monolayers (> 2000omegacm2) grown on polycarbonate filters, was studied. Model proteins were 14C-cytochrome c, 14C-ovalbumin, granulocyte-colony stimulating factor (G-CSF), 14C-bovine serum albumin (BSA), 125I-transferrin, and 14C-immunoglobulin G. Cytochrome c was extensively metabolized, as indicated by < 10% of the dose being translocated in intact form. This contrasts with 20-80% for the other model proteins studied. The flux of cytochrome c and G-CSF was symmetric in the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. By contrast, the flux of intact ovalbumin, BSA, transferrin and immunoglobulin G showed asymmetry, with the ab flux being higher by 2-5 times. There was no relationship between ab or ba fluxes and the molecular weights of these four model proteins. Since some of the proteins were translocated at much greater rates than are consistent with restricted diffusion or pinocytosis, receptor-mediated or adsorptive transcytosis may be involved.

Animals↗

PubMed2000

Functional characterization of organic cation drug transport in the pigmented rabbit conjunctiva.

PURPOSE: To characterize carrier-mediated organic cation drug transport in the rabbit conjunctiva. METHODS: The transport of [14C]guanidine, the model substrate, in the excised pigmented rabbit conjunctiva was evaluated in the modified Ussing chamber. Tetraethylammonium (TEA) transport also was investigated to determine substrate specificity. RESULTS: The apparent permeability coefficient for guanidine and TEA in the mucosal-to-serosal (ms) direction was 5.4 and 49.6 times greater than that in the serosal-to-mucosal (sm) direction, respectively. Guanidine transport in the ms (but not sm) direction revealed temperature and concentration dependency over 0.02 to 10 mM with an apparent Michaelis-Menten constant of 3.1 mM and a maximal flux of 11.4 nmol/(cm2 x h). Net guanidine transport measured at 0.1 mM across the conjunctiva was decreased by 71% or 82%, respectively, on the addition of 1 microM valinomycin (a K+ ionophore) in both bathing fluids or in a high K+ buffer in the mucosal fluid. Interestingly, net guanidine transport was reduced, rather than enhanced, by 63% upon acidifying the mucosal bathing fluid. By contrast, net guanidine transport was not affected by the serosal presence of 0.5 mM ouabain (a Na+, K+-ATPase inhibitor), by the mucosal and serosal presence of 0.1 microM monensin (a Na+ ionophore) or 0.3 microM carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP, a H+ ionophore). Guanidine transport in the ms direction was polyspecific, as indicated by the 48% to 82% inhibition by structurally diverse amines. In particular, guanidine ms transport was inhibited by the antiglaucoma drugs dipivefrine (72%), brimonidine (70%), and carbachol (78%). CONCLUSIONS: A carrier-mediated organic cation transport process appears to exist in the conjunctiva, mediating the absorption of organic amines, including certain amine-type ophthalmic drugs. This process may be driven by an inside-negative apical membrane potential difference.

Animals↗

PubMed1999

Biopharmaceutics of transmucosal peptide and protein drug administration: role of transport mechanisms with a focus on the involvement of PepT1.

Non-invasive delivery of peptide and protein drugs will soon become a reality. This is due partly to a better understanding of the endogenous transport mechanisms, including paracellular transport, endocytosis, and carrier-mediated transport of mucosal routes of peptide and protein drug administration. This paper focuses on work related to the elucidation of structure-function, intracellular trafficking, and regulation of the intestinal dipeptide transporter, PepT1.

Biological Transport↗

PubMed1999

After chromatin is SWItched-on can it be RUSHed?

Repressive chromatin must be remodeled to allow for transcriptional activation of genes in eukaryotic cells. Factors that alter chromatin structure to permit access of transcriptional activators, RNA polymerase II and the polymerase-associated general transcription factors to nucleosomal promoter sequences are as highly conserved as the basic mechanism of transcription. One group of promoter restructuring factors that perturbs chromatin in an ATP-dependent manner includes NURF, CHRAC, ACF, the SWI/SNF complex, and SWI/SNF-related proteins. Each member of this group contains a subunit homologous to the DNA-dependent ATPase; however, their individual mechanisms of action are unique. The small amount of SWI/SNF complex (100-200 copies/cell), its affiliation with a select number of inducible genes, and its interaction with the glucocorticoid and estrogen receptors, suggests the SWI/SNF complex might be preferentially targeted to active promoters. The SWI/SNF-related family of RUSH proteins which includes RUSH-1alpha and beta, hHLTF, HIP116, Zbu1, P113, and the transcription factor RUSH-1alpha isolog has been implicated as a highly conserved DNA binding site-specific ATPase.

Amino Acid Sequence↗