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F Kang

Publications and source records attributed to F Kang.

16 recordsLinked to original sources
PubMed2001

Effect of additives on the release of a model protein from PLGA microspheres.

The purpose of this study was to investigate the effect of 2 additives, poly(ethylene glycol) (PEG) 1000 and 1,2,3-tridecanoyl glycerol (tricaprin), on the physico-chemical characteristics and in vitro release of a model protein, bovine serum albumin (BSA), form poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. BSA-loaded microspheres were prepared by the double emulsion solvent evaporation method. Additives were incorporated into microspheres to modify the release of protein. The addition of PEG 1000 and tricaprin changed the surface characteristics of microspheres from smooth and nonporous to porous and dimpled, respectively. The in vitro release profiles showed that the additives significantly (P < 0.05) increased the early-stage release of BSA from microspheres.

Delayed-Action Preparations↗
PubMed2001

Mercurial sensitivity of aquaporin 1 endofacial loop B residues.

The water channel protein aquaporin-1 (AQP1) has two asparagine-proline-alanine (NPA) repeats on loops B and E. From recent structural information, these loops are on opposite sides of the membrane and meet to form a pore. We replaced the mercury-sensitive residue cysteine 189 in AQP1 by serine to obtain a mercury-insensitive template (C189S). Subsequently, we substituted three consecutive cysteines for residues 71-73 near the first NPA repeat (76-78) in intracellular loop B, and investigated whether they were accessible to extracellular mercurials. AQP1 and its mutants were expressed in Xenopus laevis oocytes, and the osmotic permeability (P(f)) of the oocytes was determined. C189S had wild-type P(f) but was not sensitive to HgCl(2). Expression of all three C189S cysteine mutants resulted in increased P(f), and all three mutants regained mercurial sensitivity. These results, especially the inhibitions by the large mercurial p-chloromercunbenzene-sulfonic acid (pCMBS) ( approximately 6A wide), suggest that residues 71-73 at the pore are accessible to extracellular mercurials. A 30-ps molecular dynamics simulation (at 300 K) starting with crystallographic coordinates of AQP1 showed that the width of the pore bottleneck (between Connolly surfaces) can vary (w(avg) = 3.9 A, sigma = 0.75; hydrated AQP1). Thus, although the pore width would be > or = 6 A only for 0.0026 of the time, this might suffice for pCMBS to reach residues 71-73. Alternative explanations such as passage of pCMBS across the AQP1 tetramer center or other unspecified transmembrane pathways cannot be excluded.

Amino Acid Motifs↗
PubMed2000

[Cultured bovine corneal epithelial cells express functional water channel].

OBJECTIVE: To investigate the water permeability (P(f)) of the cultured bovine corneal epithelial cells (CBCEC) and whether aquaporins (AQPs) were present. METHODS: Laser light-scattering system was used to determine the volume change of CBCEC from isotonic status rapidly transferred to hypotonic one, and the P(f) value was calculated. Messenger RNA obtained from CBCEC was injected into Xenopus laevis oocytes. After four days, the P(f) was determined by monitoring volume changes of oocytes upon hypoosmotic challenge (180 mOsm/L changed to 15 mOsm/L). RESULTS: CBCEC challenged with 10% hypoosmotic solution at 37 degrees C swelled rapidly; the cellular P(f) was 72 microm/s. In the oocytes injected with mRNA from CBCEC, the P(f) increased to 76 microm/s. This increase was inhibited (by 89%) by 0.3 mmol/L HgCl(2). CONCLUSION: Our results suggest that CBCEC express a typical mercurial-sensitive functional water channel in good numbers, judging from the comparatively high P(f) we measured. These begin to reopen the question of the presence and extent of an epithelial role in control of corneal hydration.

Animals↗
PubMed2000

[The protective effect of ligustrazine on optic nerve axons in rabbit eyes with continuous elevated IOP].

OBJECTIVE: To investigate the effects of ligustrazine on optic nerve axons in rabbit eyes with continuous elevated intraocular pressure (IOP). METHOD: Thirty-six NZ rabbits with continuous elevated IOP induced by injection of alpha-chymotrypsin into anterior chamber were divided into two groups by randomization: treatment and control group. Ten mg/kg ligustrazine injected into the treatment group every day for 3 weeks began on the 7(th) day after IOP elevated continuously. The changes of conjunctival microcirculation and optic nerve axonal ultrastructure were observed at different time points. RESULTS: The destruction of conjunctival microcirculation and axonal ultrastructure was milder in treatment group than that in the control group. In addition, the total number of axons and the percentage of axons in optic nerve area were higher, whereas the axonal diameter was smaller in the treatment group. CONCLUSION: It is suggested that ligustrazine protect optic nerve axons from more damage induced by elevated IOP through improving ocular microcirculation.

Animals↗
PubMed1999

Profilin promotes barbed-end actin filament assembly without lowering the critical concentration.

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1. Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin. Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin. Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4. Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4. Actin and ushers actin as a Profilin. Actin complex onto growing barbed filament ends.

Actins↗
PubMed1999

Transport of fluid by lens epithelium.

We report for the first time that cultured lens epithelial cell layers and rabbit lenses in vitro transport fluid. Layers of the alphaTN4 mouse cell line and bovine cell cultures were grown to confluence on permeable membrane inserts. Fluid movement across cultured layers and excised rabbit lenses was determined by volume clamp (37 degrees C). Cultured layers transported fluid from their basal to their apical sides against a pressure head of 3 cmH2O. Rates were (in microliter. h-1. cm-2) 3.3 +/- 0.3 for alphaTN4 cells (n = 27) and 4.7 +/- 1.0 for bovine layers (n = 6). Quinidine, a blocker of K+ channels, and p-chloromercuribenzenesulfonate and HgCl2, inhibitors of aquaporins, inhibited fluid transport. Rabbit lenses transported fluid from their anterior to their posterior sides against a 2.5-cmH2O pressure head at 10.3 +/- 0.62 microliter. h-1. lens-1 (n = 5) and along the same pressure head at 12.5 +/- 1.1 microliter. h-1. lens-1 (n = 6). We calculate that this flow could wash the lens extracellular space by convection about once every 2 h and therefore might contribute to lens homeostasis and transparency.

4-Chloromercuribenzenesulfonate↗
PubMed1999

Cultured bovine corneal epithelial cells express a functional aquaporin water channel.

PURPOSE: Given recent physiological and in situ hybridization evidence for the presence of a water channel in corneal epithelium, this study was conducted to investigate its expression and characteristics using cultured bovine corneal epithelial cells (CBCEPCs). METHODS: CBCEPCs were grown in DMEM containing 2 ng/ml fibroblast growth factor and 6% fetal bovine serum. To determine their osmotic permeability (Pf), cells were passaged onto rectangular glass coverslips, and anisotonically induced volume changes were monitored by light scattering. To investigate expression, poly(A+) RNA from CBCEPCs was injected into Xenopus laevis oocytes, and the Pf of the oocytes was determined. RESULTS: For CBCEPCs challenged with a 10% hypotonic solution at 37 degrees C, the kinetic constant of volume change was k=0.52+/-0.04 seconds(-1), and the calculated Pf 72+/-6 microm/sec (n=16). The Pf of oocytes injected with water was 14+/-1.8 microm/sec (n=4); injection with poly(A+) RNA from CBCEPCs increased Pf to 77+/-6 microm/sec (n=6). This increase in Pf was inhibited by 72% (reduced to 22+/-1 microm/sec) by 0.3 mM HgCl2 and was inhibited by 56% to 58% by coinjection with aquaporin (AQP)5 antisense oligonucleotide. CONCLUSIONS: The comparatively high Pf determined for CBCEPCs, the presence of mRNA encoding water channels, and sensitivity to mercurial agents are typical of the expression of functional water channels. The predominant message is for AQP5, although the evidence was consistent with the presence of additional water channels. These findings bring renewed support for the notion that the epithelium can contribute to corneal hydration homeostasis.

Animals↗
PubMed1998

Effects of ciprofloxacin, streptomycin, and gentamicin on rabbit corneal transendothelial electrical potential difference.

PURPOSE: A previous report suggested that high concentrations of ciprofloxacin in the anterior chamber may cause dose-dependent acute corneal decompensation. Therefore we evaluated the effect of varying concentrations of ciprofloxacin in the anterior chamber on the corneal endothelium and compared these effects with those of gentamicin and streptomycin. METHODS: We assessed endothelial transport function by determining transendothelial electrical potential differences (TEPDs) of rabbit corneas. Our control solution was bicarbonate-buffered balanced saline with glucose (BSG), to which we added ciprofloxacin (50, 100, 125, and 150 microg/ml), gentamicin (1,000 and 2,000 microg/ml), and streptomycin (196, 437, and 696 microg/ml). RESULTS: At high concentrations exceeding minimal inhibitory concentrations against 90% of common ocular isolates (MIC90), accelerated decay of TEPDs was seen with all three antibiotics. Adverse effects on TEPDs were noted at concentrations corresponding to >50 times MICs with ciprofloxacin and 40 x MICs with gentamicin, but only 2 times MICs with streptomycin. CONCLUSION: Our study shows that concentrations of ciprofloxacin, gentamicin, and streptomycin below or equal to their MIC90 levels do not adversely affect endothelial transport function in a rabbit model.

Animals↗
PubMed1998

Sodium, potassium, two chloride cotransport in corneal endothelium: characterization and possible role in volume regulation and fluid transport.

PURPOSE: To search for membrane transporter proteins that could contribute to volume regulation and fluid transport by corneal endothelium. As an initial step, the authors have focused on Na+-K+-2Cl- cotransporters. METHODS: Bovine corneal endothelial cells were cultured to confluence. 86Rubidium was used as a tracer for K+ uptake determinations; uptake values were normalized per milligram of cell protein. RESULTS: Three components of K+ uptake were characterized: ouabain (1 mM) sensitive, bumetanide (0.1 mM) sensitive, and ouabain-bumetanide insensitive. Both the ouabain-sensitive and bumetanide-sensitive components increased in the presence of 26.2 mM HCO3-; 0.5 mM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid abolished this increase. The bumetanide-sensitive component was completely inhibited in the absence of Na+ or Cl-. This component was increased 33% by a 33% hypertonic solution and was decreased 38% by a 33% hypotonic solution. The protein kinase C activator phorbol 12-myristate 13-acetate decreased the activity of the cotransporter, whereas forskolin, in the presence of isobutylmethylxanthine, decreased it. Calyculin A (100 nM), an inhibitor of phosphatases 1 and 2a, produced a large (97%) activation of this component. CONCLUSIONS: These results provided for the first time conclusive evidence for the presence of a Na+-K+-2Cl- cotransporter in corneal endothelium and of its possible involvement in volume-regulatory processes in these cells. Given the uptake values reported here, such cotransporter could contribute significantly to electrolyte transport and hence to fluid transport across this preparation.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Vinculin proteolysis unmasks an ActA homolog for actin-based Shigella motility.

To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotein (VASP) (Brindle, N.P.J., M. R. Hold, J.E. Davies, C.J. Price, and D.R. Critchley. 1996. Biochem. J. 318:753-757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.

Actins↗
PubMed1997

Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): implications for actin-based Listeria motility.

Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.

Actins↗
PubMed1997

Macular holes: migratory gaps and vitreous as obstacles to glial closure.

PURPOSE: Retinal glia may play an important role in the closure of macular holes. This in vitro study examines whether and how the specific pathoanatomy, including foveal eversion and foveal vitreous, may interfere with glial closure of macular holes. METHODS: Culture dishes used to grow glial cells were modified by the placement of slopes, vertical steps, and gaps to mimic the in vivo migratory surface in and surrounding macular holes. In separate experiments, defects were made in a rodent glial monolayer. These defects were exposed to hyaluronic acid (HA) and to rabbit (RV) and bovine (BV) vitreous gel. The migratory behavior and completeness of closure of defects were compared to controls. RESULTS: As expected, glial cells migrated further and in greater numbers on a smooth surface. Slopes and steps were moderate obstacles to migration; gaps in the surface were absolute obstacles. HA modified the pattern of adhesion of cells at the bottom of defects. Defects in the glial monolayer were repaired in 5-7 days. Compared to these controls, repair was inhibited by 11% (n.s.), 28% (P = 0.02), and 58% (P = 0.004) after direct exposure of defects to HA, RV and BV, respectively. CONCLUSION: The elevated and everted margins of macular holes represent slope, step, and gap-like obstacles to the migration of glial cells and hence to the healing of defects. The defect allows extension of extracellular matrix into it and the subretinal space. Our results indicate that gaps in the migratory surface caused and aggravated by eversion and the presence of vitreous present obstacles to glial migration and closure of macular holes.

Animals↗
PubMed1997

[The toxicity of gentamicin on corneal cells in culture].

OBJECTIVE: To determine the safe local concentration of gentamicin. METHODS: The authors studied the toxicity of gentamicin (no preservative) on 3 types of human corneal cell in culture with different concentrations. RESULTS: There was no toxic effect on the corneal epithelium, stroma and endothelium with gentamicin 1 mg. per milliliter in 48 h. in vitro, while the concentration was up to 2 mg. or 4 mg. per milliliter, all types of corneal cells appeared cytopathic effect (CPE), the toxicity of gentamicin to the corneal cells is in direct proportion to its concentration and time term of incubation, and the toxicity was the greatest on endothelium, slightest on stroma: CONCLUSION: It is suggested that the safe dosage of local concentration of gentamicin be 1 mg. per milliliter in human eyes.

Cells, Cultured↗
PubMed1996

Platelet activating factor inhibits fluid transport by corneal endothelium.

PURPOSE: Given reports of corneal edema after endothelial exposure to platelet activating factor (PAF), the authors have investigated whether PAF can affect the function of corneal endothelium in vitro. METHODS: The endothelial side of deepithelialized rabbit corneas was perfused with BSS+ and test agents: PAF, its inactive receptor ligand analog Lyso-PAF, and its antagonist BN52021. Stromal thickness was determined by specular microscopy. Translayer-specific electrical resistance (rho) was measured in cultured bovine corneal endothelial cells grown on permeable substrates at 36.5 degrees C. RESULTS: Control corneas perfused with BSS+ or with BSS+ containing Lyso-PAF swelled at a very slow rate (6.2 +/- 0.1, and 7.9 +/- 0.2 microns/hour, respectively). Corneas exposed to PAF swelled appreciably faster and at rates that were a saturable function of PAF (K(m), 2.1 microM); maximal rates of swelling were < 20 microns/hour, indicating no appreciable damage to intercellular junctions. BN52021 prevented PAF-induced swelling (Ki, 1.1 microM). PAF led also to a decrease in rho (from 42.8 +/- 1.4 to 24.5 +/- 0.6 omega cm2 in 1 hour; 46.8 +/- 1.5 to 38.3 +/- 1.4 omega cm2 in control layers; and 43.0 +/- 1.2 to 30.8 +/- 1.6 omega cm2 in layers exposed to PAF+BN52021). Such rho changes are consistent with swelling of intercellular spaces. CONCLUSIONS: Results suggest that PAF inhibits transendothelial fluid transport on binding to an endothelial cell receptor for it; continuous stimulation of a PAF-induced signaling cascade may lead to such inhibition. From these and other results, fluid transport might result from cascades activating sequentially basolateral and apical transporters or channels.

Animals↗
PubMed1993

Production of interferon alpha A by Escherichia coli W3110 (pEC901).

The maximum specific growth rate, saturation constant, maintenance coefficient and growth yield for E. coli W3110 (pEC901) and the host, W3110, were estimated through continuous cultivation of the two strains. The growth rate of the transformant was lower than that of the host, while its maintenance metabolism increased due to the existence of plasmids pEC901. Both the plasmid stability and the expression level of interferon increased with the increase in dilution rate. In fed-batch culture, the growth rate of W3110 (pEC901) was maintained at a high level by controlling the feeding rate of glucose, and the interferon titer reached 2.5 x 10(10) u/l, which was greatly improved compared with those reached in shaker flask cultures.

Escherichia coli↗