Survival and integration of tissue-engineered corneal stroma in a model of corneal ulcer

Tissue-engineered replacement of diseased or damaged tissue has become a reality for some types of tissue, such as skin and cartilage. Tissue-engineered corneal stroma represents a promising concept to overcome the limitations of cornea replacement with allograft. In this study, porcine cornea was decellularized by a series of extraction methods, and the in vivo biocompatibility of the scaffold was measured subcutaneously in rabbits (n = 8). These were not acutely rejected and no abscesses were observed by hematoxylin and eosin staining at the 8th week, indicating that the scaffolds had good biocompatibility. To investigate the potential value of clinical applications, rabbit stromal keratocytes were implanted onto decellularized scaffolds to fabricate tissue-engineered corneal stroma. Allograft, tissue-engineered corneal stroma, or scaffolds were implanted into a model of corneal ulcer. The survival and reconstruction of corneal transplantation were morphologically evaluated by light and electron microscopy until the 32nd week after implantation. Experiments involving transplantation indicated that the epithelial and stromal defect healed quickly, with improvement in corneal clarity. The integration of the graft was accompanied by neurite ingrowth from the host tissue. By 16 weeks after transplantation, the cornea had gradually regained an intact state similar to that of normal cornea. Our results demonstrate that the tissue-engineered corneal stroma with allogenetic cells is a promising therapeutic method for corneal injury. This study was supported by the Nature Science Foundation of China (project no. 30572046) and the Development of High and New Science and Technology (863 Project) of China (2002AA205041, 2005AA205241).     

Prey capture and accommodation in the sandlance, Limnichthyes fasciatus (Creediidae; Teleostei)

The eyes of the sandlance, Limnichthyes fasciatus (Creediidae, Teleostei) move independently and possess a refractive cornea, a convexiclivate fovea and a non-spherical lens giving rise to a wide separation of the nodal point from the axis of rotation of the eye much like that of a chameleon. To investigate this apparent convergence of the visual optics in these phylogenetically disparate species, we examine feeding behaviour and accommodation in the sandlance with special reference to the possibility that sandlances use accommodation as a depth cue to judge strike length. Frame-by-frame analysis of over 2000 strikes show a 100% success rate. Explosive strikes are completed in 50 ms over prey distances of four body lengths. Close-up video confirms that successful strikes can be initiated monocularly (both normally and after monocular occlusion) showing that binocular cues are not necessary to judge the length of a strike. Additional means of judging prey distance may also be derived from parallax information generated by rotation of the eye as suggested for chameleons. Using photorefraction on anaesthetised sandlances, accommodative changes were induced with acetylcholine and found to range between 120 D and 180 D at a speed of 600–720 D s⁻¹. The large range of accommodation (25% of the total power) is also thought to be mediated by corneal accommodation where the contraction of a unique cornealis muscle acts to change the corneal curvatures. Accepted: 8 December 1999     

Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse β-defensins-2, -3 and -4 (mBD2–4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that β-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.     

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol⁻¹. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol⁻¹ at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h. Received: 12 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

Determination of the true intraocular pressure and modulus of elasticity of the human cornea in vivo

The purpose of this study was to determine the true intraocular pressure and modulus of elasticity of the human cornea in vivo. The cornea was modeled as a shell, and the equations for the deformations of a shell due to applanating and intraocular pressures were combined to model the behavior of the cornea during applanation tonometry. At certain corneal dimensions called the calibration dimensions, the applanating and intraocular pressures are considered to be equal. This relationship was used to determine the modulus of elasticity of the cornea and the relationship between the applanating and intraocular pressures. The true intraocular pressure (IOPT) was found to be related to Goldmann’s applanating pressure (IOPG) as (IOPT = IOPG/K, where K is a correction factor. For the calibration corneal thickness of 0.52 mm, the modulus of elasticity E in MPa of the human cornea was found to be related to the true intraocular pressure IOPT in mmHg as E = 0.0229IOPT. The generalization of the Imbert—Fick law that takes into account the effect of corneal dimensions and stiffness was found to be given by IOPT = 73.5W/(K A), where W is the applanating weight in gf (gram force) and A is the applanated area in mm². The calculated true intraocular pressure and modulus of elasticity were found to agree with published experimental results. The mathematical model developed may therefore be used to improve results from applanation tonometry and to estimate the mechanical property of the cornea in vivo.     

Xenogeneic Acellular Conjunctiva Matrix as a Scaffold of Tissue-Engineered Corneal Epithelium

Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface.     

Production of sophorolipids from whey

Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1′,4′′-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFT^min 39 mN m⁻¹, CMC 130 mg l⁻¹), water solubilities (2–3 g l⁻¹) and low cytotoxicities (LC₅₀ 300–700 mg l⁻¹). In contrast, purified sophorolipids were more surface active (SFT^min 36 mN m⁻¹, CMC 10 mg l⁻¹), less water soluble (max. 70 mg l⁻¹) and showed stronger cytotoxic effects (LC₅₀ 15 mg l⁻¹). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect. Received: 23 February 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

Effect of hydroxypropyl beta cyclodextrin complexation on aqueous solubility,stability, and corneal permeation of acyl ester prodrugs of ganciclovir

The purpose of the study was to investigate the effect of hydroxypropyl beta cyclodextrin (HPβCD) on aqueous solubility, stability, and in vitro corneal permeation of acyl ester prodrugs of ganciclovir (GCV). Aqueous solubility and stability of acyl ester prodrugs of Ganciclovir (GCV) were evaluated in pH 7.4 isotonic phosphate buffer solution (IPBS) in the presence and absence of HPβCD. Butyryl cholinesterase-mediated enzymatic hydrolysis of the GCV prodrugs was studied using various percentage w/v HPβCD. In vitro corneal permeation of GCV and its prodrugs (with and without 5% HPβCD) across isolated rabbit cornea was studied using side-by-side diffusion cells. HPβCD-prodrug complexation was of the A_L type with values for complexation constants ranging between 12 and 108 M⁻¹. Considerable improvement in chemical and enzymatic stability of the GCV prodrugs was observed in the presence of HPβCD. The stabilizing effect of HPβCD was found to depend on the degree of complexation and the degradation rate of prodrug within the complex. Five percent w/v HPβCD was found to enhance the corneal permeation of only the most lipophilic prodrug GCV dibutyrate (2.5-fold compared with 0% HPβCD). All other prodrugs showed little or no difference in transport in the presence of 5% w/v HPβCD. Agitation in the donor chamber largely influenced the transport kinetics of GCV dibutyrate across cornea. Results indicate the presence of an unstirred aqueous diffusion layer at the corneal surface that restricts the transport of the highly lipophilic GCV dibutyrate prodrug. HPβCD improves corneal permeation by solubilizing the hydrophobic prodrug and delivering it across the mucin layer at the corneal surface.     

Influence of the Selected Antioxidants on the Stability of the Celsior Solution Used for Perfusion and Organ Preservation Purposes

The purpose of the following research was to improve the original Celsior solution in order to obtain a higher degree of stability and effectiveness. The solution was modified by the addition of selected antioxidants such as vitamin C, cysteine, and fumaric acid in the following concentrations: 0.1, 0.3, and 0.5 mmol/l. The solution’s stability was estimated using an accelerated stability test based on changes in histidine concentrations in the solution using Pauly’s method for determining concentrations. Elevated temperatures, the factor accelerating substances’ decomposition reaction rate, were used in the tests. The research was conducted at four temperatures at intervals of 10°C: 60 ± 0.2°C, 70 ± 0.2°C, 80 ± 0.2°C, and 90 ± 0.2°C. It was stated that the studied substances’ decomposition occurred in accordance with the equation for first-order reactions. The function of the logarithmic concentration (log%C) over time was revealed to be rectilinear. This dependence was used to determine the kinetics of decomposition reaction rate parameters (the rate constant of decomposition k, activation energy E _a, and frequency factor A). On the basis of these parameters, the stability of the modified solution was estimated at +5°C. The results obtained show that the proposed antioxidants have a significant effect on lengthening the Celsior solution’s stability. The best results were reached when combining two antioxidants: vitamin C and cysteine in 0.5 mmol/l concentrations. As a result, the Celsior solution’s stability was lengthened from 22 to 299 days, which is 13.5 times. Vitamin C at a concentration of 0.5 mmol/l increased the solution’s stability by 5.2 times (t ₉₀ = 115 days), cysteine at a concentration of 0.5 mmol/l caused a 4.4 times stability increase (t ₉₀ = 96 days), and fumaric acid at a concentration of 0.5 mmol/l extended the stability by 2.1 times (t ₉₀ = 48 days) in relation to the original solution.     

Noscapinoids with anti-cancer activity against human acute lymphoblastic leukemia cells (CEM): a three dimensional chemical space pharmacophore modeling and electronic feature analysis

We have identified a new class of microtubule-binding compounds—noscapinoids—that alter microtubule dynamics at stoichiometric concentrations without affecting tubulin polymer mass. Noscapinoids show great promise as chemotherapeutic agents for the treatment of human cancers. To investigate the structural determinants of noscapinoids responsible for anti-cancer activity, we tested 36 structurally diverse noscapinoids in human acute lymphoblastic leukemia cells (CEM). The IC₅₀ values of these noscapinoids vary from 1.2 to 56.0 μM. Pharmacophore models of anti-cancer activity were generated that identify two hydrogen bond acceptors, two aromatic rings, two hydrophobic groups, and one positively charged group as essential structural features. Additionally, an atom-based quantitative structure–activity relationship (QSAR) model was developed that gave a statistically satisfying result (R ² = 0.912, Q ² = 0.908, Pearson R = 0.951) and effectively predicts the anti-cancer activity of training and test set compounds. The pharmacophore model presented here is well supported by electronic property analysis using density functional theory at B3LYP/3-21*G level. Molecular electrostatic potential, particularly localization of negative potential near oxygen atoms of the dimethoxy isobenzofuranone ring of active compounds, matched the hydrogen bond acceptor feature of the generated pharmacophore. Our results further reveal that all active compounds have smaller lowest unoccupied molecular orbital (LUMO) energies concentrated over the dimethoxy isobenzofuranone ring, azido group, and nitro group, which is indicative of the electron acceptor capacity of the compounds. Results obtained from this study will be useful in the efficient design and development of more active noscapinoids.     

Differential expression of the keratan sulphate proteoglycan,keratocan, during chick corneal embryogenesis

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the connective tissue matrix of the cornea of the eye, where they are believed to have functional roles in tissue organisation and transparency. Keratocan, is one of the three KS PGs expressed in cornea, and is the only one that is primarily cornea-specific. Work with the developing chick has shown that mRNA for keratocan is present in early corneal embryogenesis, but there is no evidence of protein synthesis and matrix deposition. Here, we investigate the tissue distribution of keratocan in the developing chick cornea as it becomes compacted and transparent in the later stages of development. Indirect immunofluorescence using a new monoclonal antibody (KER-1) which recognises a protein epitope on the keratocan core protein demonstrated that keratocan was present at all stages investigated (E10–E18), with distinct differences in localisation and organisation observed between early and later stages. Until E13, keratocan appeared both cell-associated and in the stromal extracellular matrix, and was particularly concentrated in superficial tissue regions. By E14 when the cornea begins to become transparent, keratocan was located in elongate arrays, presumably associated along collagen fibrils in the stroma. This fibrillar label was still concentrated in the anterior stroma, and persisted through E15–E18. Presumptive Bowman’s layer was evident as an unlabelled subepithelial zone at all stages. Thus, in embryonic chick cornea, keratocan, in common with sulphated KS chains in the E12–E14 developmental period, exhibits a preferential distribution in the anterior stroma. It undergoes a striking reorganisation of structure and distribution consistent with a role in relation to stromal compaction and corneal transparency. E. Claire Gealy and Briedgeen C. Kerr were joint first authors.     

DFT and MP2 investigations of <Emphasis Type="SmallCaps">L</Emphasis>-proline and its hydrated complexes

A theoretical study of L-proline-nH₂O (n = 1–3) has been performed using the hybrid DFT-B3LYP and MP2 methods together with the 6-311++G(d,p) basis set. The results show that the P2 conformer is energetically favorable when forming a hydrated structure, and the hydration of the carboxyl group leads to the greatest stability. For hydrated complexes, the adiabatic and vertical singlet–triplet excitation energies tend to decrease with the addition of water molecules. The hydration energy indicates that in the hydrated complexes the order of stability is: binding site 2 > binding site 1 > binding site 3, and binding site 12 > binding site 23 > binding site 13. As water molecules are added, the stabilities of these hydrated structures gradually increase. In addition, an infrared frequency analysis indicated that there are some differences in the low-frequency range, which are mainly dominated by the O–H stretching or bending vibrations of different water molecules. All of these results should aid our understanding of molecular behavior and provide reference data for further studies of biological systems.     

Sivelestat relaxes vascular smooth muscle contraction in human gastric arteries

Sivelestat sodium hydrate (sivelestat) is a novel synthetic drug and specific inhibitor of neutrophil elastase that has been approved in Japan as a treatment for acute lung injury associated with systemic inflammatory response syndrome. It is important to determine how sivelestat affects hemodynamics and the regulatory mechanisms of vascular smooth muscle (VSM). We recently found that sivelestat relaxes porcine coronary artery VSM via selective inhibition of Ca²⁺ sensitization induced by a receptor agonist without affecting the normal Ca²⁺-induced contraction. Although sivelestat relaxes porcine artery, its effects on human artery are unknown; therefore, the purpose of the present study was to assess the effects of sivelestat on human artery. In the present study, sivelestat induced concentration-dependent (1 × 10⁻⁶ to 3 × 10⁻⁴ M) vasorelaxation in U46619 (1 nM) and sphingosylphosphorylcholine (SPC) (30 mM)-precontracted human gastric artery with or without endothelium, but sivelestat did not induce vasorelaxation in conditions of high K⁺ (40 mM) depolarization. Sivelestat inhibited VSM contraction by an agonist and SPC, and it did not affect Ca²⁺-induced normal physiologic contraction.     

Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor

A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-l sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l⁻¹ h⁻¹. This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH. Received: 16 November 1998 / Received revision: 28 March 1999 / Accepted: 9 April 1999     

Biocompatibility evaluation of bioprinted decellularized collagen sheet implanted in vivo cornea using swept‐source optical coherence tomography

Corneal transplantation by full‐thickness penetrating keratoplasty with human donor tissue is a widely accepted treatment for damaged or diseased corneas. Although corneal transplantation has a high success rate, a shortage of high‐quality donor tissue is a considerable limitation. Therefore, bioengineered corneas could be an effective solution for this limitation, and a decellularized extracellular matrix comprises a promising scaffold for their fabrication. In this study, three‐dimensional bioprinted decellularized collagen sheets were implanted into the stromal layer of the cornea of five rabbits. We performed in vivo noninvasive monitoring of the rabbit corneas using swept‐source optical coherence tomography (OCT) after implanting the collagen sheets. Anterior segment OCT images and averaged amplitude‐scans were acquired biweekly to monitor corneal thickness after implantation for 1 month. The averaged cornea thickness in the control images was 430.3 ± 5.9 μm, while the averaged thickness after corneal implantation was 598.5 ± 11.8 μm and 564.5 ± 12.5 μm at 2 and 4 weeks, respectively. The corneal thickness reduction of 34 μm confirmed the biocompatibility through the image analysis of the depth‐intensity profile base. Moreover, hematoxylin and eosin staining supported the biocompatibility evaluation of the bioprinted decellularized collagen sheet implantation. Hence, the developed bioprinted decellularized collagen sheets could become an alternative solution to human corneal donor tissue, and the proposed image analysis procedure could be beneficial to confirm the success of the surgery.      

Comparing the use of leaf and cambium tissue in a single genetic study of tropical trees

Amplified fragment length polymorphisms (AFLPs) are a useful molecular tool for studying species with little available genetic information; however, since universal primers are used contaminant DNA from non-target organisms may also be amplified. Cambium tissue may contain fewer biotic contaminants or plant defense chemicals, than the more commonly used leaf tissue, and therefore be more suitable for use as a source of DNA when using universal primers. On the other hand, cambium tissue can be difficult to identify, yields low DNA and requires the bark of the tree to be damaged, thereby increasing the risk of introducing disease. We show that within two tropical tree species, there are few differences between AFLP profiles obtained from either cambium or leaf tissue from the same tree. We studied 50 Brosimum alicastrum individuals at 119 AFLP loci and 40 Swietenia macrophylla individuals at 112 AFLP loci. The matrix of S?rensen similarity indices between individual AFLP profiles for cambium samples was strongly correlated to the matrix for leaf samples in each species (Mantel test; B. alicastrum r = 0.815, P < 0.001; S. macrophylla r = 0.895, P < 0.001). The phylogenetic relationship between the trees studied did not differ dependent on tissue type used and therefore shows that both tissues can be used within a single study without introducing substantial error.     

Hydrogen-Related Enhancement of In Vivo Antioxidant Ability in the Brain of Rats Fed Coral Calcium Hydride

This study explored the effect of coral calcium hydride (CCH) on rat intrahippocampal antioxidant ability by measuring the PCAM nitroxide radical decay ratio when CCH was (a) co-perfused into the hippocampus and (b) fed orally to the rats for 4 weeks under a freely moving state. Estimation of the in vivo antioxidant effect was obtained by administration of the blood–brain barrier-permeable PCAM nitroxide radical and the measured PCAM radical decay ratio then correlated to the amount of antioxidant in the brain using electron spin resonance (ESR) spectroscopy combined with microdialysis. The half-life periods of PCAM in rats treated with CCH in both the co-perfusion and orally fed groups were significantly shorter compared to the control group. These results clarify the mechanism that CCH may exert antioxidant activity by significantly enhancing the basal endogenous antioxidant ability in the hippocampus through a synergistic effect with α-tocopherol and ascorbic acid.     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

Quercetin in Lyotropic Liquid Crystalline Formulations: Physical, Chemical and Functional Stability

The purpose of this study was to develop a lyotropic liquid crystalline formulation using the emulsifier vitamin E TPGS and evaluate its behavior after incorporation of a flavonoid, quercetin. The physical (macro and microscopic), chemical (determination of quercetin content by the HPLC method) and functional (determination of quercetin antioxidant activity by DPPH^• assay) stability of the lamellar liquid crystalline formulation containing flavonoid was evaluated when stored at 4 ± 2 °C; 30 ± 2 °C/70 ± 5% RH (relative humidity) and 40 ± 2 °C/70 ± 5% RH during 12 months. The lamellar liquid crystalline structure of the formulation was maintained during the experiment, however chemical and functional stability results showed a great influence of the storage period in all conditions tested. A significant decrease in quercetin content (approximately 40%) was detected during the first month of storage and a similar significant loss in antioxidant activity was detected after 6 months. The remaining flavonoid content was unchanged during the final 6 months of the experimental period. The results suggest possible interactions between quercetin and the liquid crystalline formulation, which could inhibit or reduce the quercetin activity incorporated in the system. In conclusion, the present study demonstrated that incorporation of quercetin (1%) did not affect the liquid crystalline structure composed of vitamin E TPGS/IPM/PG–H₂O (1:1) at 63.75/21.25/15 (w/w/w). Nevertheless, of the total quercetin incorporated in the system only 60% was free to act as an antioxidant.     

Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999