The variation in transparency of amniotic membrane used in ocular surface regeneration

Background/aims: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. Methods: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. Results: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. Conclusion: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency.

Differentiation status of limbal epithelial cells cultured on intact and denuded amniotic membrane before and after air-lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under ‘air-lifting’ conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting and quantitative PCR. Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane. On denuded amniotic membrane the proportion of differentiated cells remained unaltered following airlifting. Within cells grown on intact amniotic membrane, however, the number of differentiated cells increased significantly following air-lifting. These results have important implications for both basic and clinical research. Firstly, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Secondly, these findings serve as a warning to clinicians that the affect of amniotic membrane on transplantable cells is not fully understood; the use of intact or denuded amniotic membrane can produce different results in terms of the amount of differentiation, once cells are exposed to the air.

Topical delivery of a naproxen-dithranol co-drug: in vitro skin penetration, permeation, and staining

Purpose This work probed the topical delivery and skin-staining properties of a novel co-drug, naproxyl-dithranol (Nap-DTH), which comprises anti-inflammatory (naproxen) and anti-proliferative (dithranol) moieties. Method Freshly excised, full-thickness porcine ear skin was dosed with saturated solutions of the compounds. After 24 h, the skin was recovered and used to prepare comparative depth profiles by the tape-stripping technique and to examine the extent of skin staining. Results Depth profiles showed that Nap-DTH led to a 5-fold increase in drug retention in the skin compared to dithranol. The application of Nap-DTH also demonstrated improved stability, resulting in lower levels of dithranol degradation products in the skin. Furthermore, significantly less naproxen from hydrolysed Nap-DTH permeated into the receptor phase compared to naproxen when applied alone (0.08 ± 0.03 nmol cm-² and 180 ± 60 nmol cm-², respectively). Moreover, the reduced staining of the skin was very apparent for Nap-DTH compared to dithranol. Conclusions Topical delivery of Nap-DTH not only improves the delivery of naproxen and dithranol, but also reduces unwanted effects of the parent moieties, in particular the skin staining, which is a major issue concerning the use of dithranol.

A further study of the recovery and purification of surfactin from fermentation broth by membrane filtration

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, a two-step membrane filtration process was developed using a lab scale tangential flow filtration (TFF) unit with 10 kDa MWCO regenerated cellulose (RC) and polyethersulfone (PES)membranes at three different transmembrane pressure (TMP) of 1.5 bar, 2.0 bar and 2.5 bar. Two modes of filtrations were studied, with and without cleaning of membranes prior to UF-2. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effectively retained by the membrane in UF-2. Flux of permeates, rejection coefficient (R) of surfactin and proteinwere measured during the filtrations. Overall the three different TMPs applied have no significant effect in the filtrations and PES is the more suitable membrane to selectively separate surfactin from fermentation broth, achieving high recovery and level of purity. In addition this two-step UF process is scalable for larger volume of samples without affecting the original functionality of surfactin, although membranes permeability can be affected due to exposure to methanolic solution used in UF-2.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Pervaporation dehydration of isopropyl alcohol with NaY zeolite incorvorated hybrid membranes

With a solution technique, NaY zeolite incorporated, tetraethylorthosilicate-crosslinked poly(vinyl alcohol) membranes were prepared. The resulting membranes were tested for their ability to separate isopropyl alcohol/water mixtures by pervaporation in the temperature range of 30-50 degrees C. The effects of the zeolite content and feed composition on the pervaporation performance of the membranes were investigated. The experimental results demonstrated that both flux and selectivity increased simultaneously with increasing zeolite content in the membranes. This was explained on the basis of the enhancement of hydrophilicity, selective adsorption, and establishment of a molecular sieving action attributed to the creation of pores in the membrane matrix. The membrane containing 15 mass % zeolite exhibited the highest separation selectivity of 3991 with a flux of 5.39 X 10(-2) kg/m(2) h with 10 mass % water in the feed at 30 degrees C. The total flux and flux of water were close to each other for almost all the studied membranes, and this suggested that the membranes could be used effectively to break the azeotropic point of water/isopropyl alcohol mixtures to remove a small amount of water from isopropyl alcohol. From the temperature-dependent diffusion and permeation values, the Arrhenius activation parameters were estimated. The activation energy values obtained for water were significantly lower than those for isopropyl alcohol, and this suggested that the developed membranes had a higher separation efficiency for water/isopropyl alcohol systems. The activation energy values for total permeation and water permeation were found to be almost the same for all the membranes, and this signified that coupled transport was minimal because of the highly selective nature of the membranes. Positive heat of sorption values were observed in all the membranes, and this suggested that Henry's mode of sorption was predominant. (c) 2008 Wiley Periodicals, lnc.

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity. (C) 2004 Wiley Periodicals, Inc.

Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from Kluyveromyces lactis: studies on batch and continuous UF membrane-fitted bioreactors

A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact(R) L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40degreesC and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40degreesC, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAEPAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal(R)GOS). (C) 2004 Wiley Periodicals, Inc.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbarnyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 mu m ODS (C-18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min(-1) and the column temperature was maintained at 30 degrees C Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32 +/- 1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15 +/- 0.1 cm(2). The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1 % v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 mu g ml(-1). The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) < 12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <=-5.60 and <=-8.00, respectively. Using this assay, it was found that GL-HCI permeates through human skin with a flux 1.497 +/- 0.42 mu g cm(-2) h(-1), a permeability coefficient of 5.66 +/- 1.6 x 10(-6) cm h(-1) and with a lag time of 10.9 +/- 4.6 h. (c) 2005 Elsevier B.V. All rights reserved.

Permeation of bioactive constituents from Arnica montana preparations through human skin in-vitro

This study investigated and characterised transdermal permeation of bioactive agents from a topically applied Arnica montana tincture. Permeation experiments conducted over 48 h used polyclimethylsiloxane (silastic) and human epidermal membranes mounted in Franz-type diffusion cells with a methanol-water (50:50 v/v) receptor fluid. A commercially available tincture of A. montana L. derived from dried Spanish flower heads was a donor solution. Further donor solutions prepared from this stock tincture concentrated the tincture constituents 1, 2 and 10 fold and its sesquiterpene lactones 10 fold. Permeants were assayed using a high-performance liquid chromatography method. Five components permeated through silastic membranes providing peaks with relative retention factors to an internal standard (santonin) of 0.28, 1.18, 1.45, 1.98 and 2.76, respectively. No permeant was detected within 12 h of applying the Arnica tincture onto human epidermal membranes. However, after 12 h, the first two of these components were detected. These were,shown by Zimmermann reagent reaction to be sesquiterpene lactones and liquid chromatography/diode array detection/mass spectrometry indicated that these two permeants were 11,13-dihydrohelenalin (DH) analogues (methacrylate and tiglate esters). The same two components were also detected within 3 h of topical application of the 10-fold concentrated tincture and the concentrated sesquiterpene lactone extract.