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Descemet's membrane endothelial keratoplasty (DMEK) may provide fast visual rehabilitation in the therapy of corneal endothelial disorders. However, due to shortage of donated corneas, how to construct a corneal endothelial substitute with powerful functions that can be used for DMEK is still unsolved. Herein, we introduced the method of corneal crosslinking (CXL) and conjugated the components of native Descemet's membrane (DM) to improve the mechanical properties and the biocompatibility of denuded amniotic membrane (dAM), further assessed their effects on cell adhesion, proliferation, YAP translocation, and metabolic activity in human corneal endothelial (HCE) cells. Using modified crosslinked dAM (mcdAM) and non-transfected HCE cells, we constructed a tissue-engineered HCE (TE-HCE) and evaluated its functions in cat and monkey models as well. Our results showed that the mechanical properties of mcdAM were improved effectively by CXL, and the adhesion, proliferation, and YAP translocation of HCE cells were dose-dependently improved after ECM modification. The combination of 0.01 mg/mL laminin with 0.1 mg/mL fibronectin showed the highest efficacy. Then, the TE-HCE was constructed in vitro, with a high density of 3612 ± 243 cells/mm

Tissue-engineered cornea endothelial sheets (TECES), created using a biocompatible thin and transparent carrier with corneal endothelial cells, could alleviate the shortage of donor corneas and provide abundant functional endothelial cells. In our previous clinical trials, the effectiveness and safety of the acellular porcine corneal stroma (APCS) applied in lamellar keratoplasty have been confirmed. In this study, we optimized the method to cut APCS into multiple 20 μm ultrathin lamellae by a cryostat microtome and investigated the feasibility of TECES by seeding rabbit corneal endothelial cells (RCECs) on ultrathin APCS. Cell adhesion, proliferation, and functional gene expression of RCECs on tissue-culture plastic and APCS of different thicknesses were compared. The results indicated that ultrathin lamellae were superior in increasing cell viability and maintaining cell functions. Analyzing with histology, electron microscopy, and immunofluorescence, we found that RCECs cultured on 20 μm ultrathin APCS for 5 days grew into a confluent monolayer with a density of 3726 ± 223 cells/mm

Human corneal endothelial cells (HCECs) play pivotal roles in maintaining corneal transparency, integrity, and thickness. However, their proliferation ceases in a mature human corneal endothelium. ...

Pranoprofen (PPF) is a wildly used anti-inflammatory ophthalmic drug. It was reported that PPF could decrease early epithelialization of scrape wounds in rabbit cornea and could reduce cell activities of cultured human corneal endothelial cells. However, effects of PPF on corneal stromal cells playing important roles in corneal wound healing remain unknown. In this study,in vitro model of cultured human corneal stomal (HCS) cells and in vivo model of rabbit corneas were used to investigate the effects and underlying mechanisms of PPF. Our findings showed that high concentrations of PPF treatment (0.1 % to 0.0125 %) caused limited chromatin condensation and quickly decreased cell viability that was proved to initiate necroptosis in HCS cells through activating receptor interacting protein kinase (RIPK) and mixed lineage kinase domain-like (MLKL). While low concentrations of PPF treatment (0.00625 %) induced DNA fragmentation, apoptotic body formation, ROS generation, activation of caspases and increase in cytoplasmic content of Bad, Bax and cytoplasmic cytochrome c that suggested apoptosis happened through ROS-mediated caspase-dependent and caspase-independent pathways. Studies of rabbit corneas treated with 0.1 % PPF (the clinical concentration) showed that PPF could induce apoptosis of rabbit corneal stromal cells. This work would be helpful for better understanding cytotoxic effects PPF on human corneal cells.

To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing ...

Aim To reveal the cytotoxicity and related mechanisms of gatifloxacin (GFX) to stromal fibroblasts (SFs) in vitro. Methods SFs were treated with GFX at different concentrations (0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine (PS) externalization, and mitochondrial transmembrane potential (MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins. Results Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2 (Bcl-2) and B-cell leukemia-XL (Bcl-XL), and upregulate Bcl-2 assaciated X protein (Bax), Bcl-2-associated death promoter (Bad), Bcl-2 interacting domain (Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs. Conclusion The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.

The introduction of intracameral anaesthesia by injection of lidocaine has become popular in cataract surgery for its inherent potency, rapid onset, tissue penetration, and efficiency. However, intracameral lidocaine causes corneal thickening, opacification, and corneal endothelial cell loss. Herein, we investigated the effects of lidocaine combined with sodium ferulate, an antioxidant with antiapoptotic and anti-inflammatory properties, on lidocaine-induced damage of corneal endothelia with in vitro experiment of morphological changes and cell viability of cultured human corneal endothelial cells and in vivo investigation of corneal endothelial cell density and central corneal thickness of cat eyes. Our finding indicates that sodium ferulate from 25 to 200 mg/L significantly reduced 2 g/L lidocaine-induced toxicity to human corneal endothelial cells, and 50 mg/L sodium ferulate recovered the damaged human corneal endothelial cells to normal growth status. Furthermore, 100 mg/L sodium ferulate significantly inhibited lidocaine-induced corneal endothelial cell loss and corneal thickening in cat eyes. In conclusion, sodium ferulate protects human corneal endothelial cells from lidocaine-induced cytotoxicity and attenuates corneal endothelial cell loss and central corneal thickening of cat eyes after intracameral injection with lidocaine. It is likely that the antioxidant effect of sodium ferulate reduces the cytotoxic and inflammatory corneal reaction during intracameral anaesthesia.

Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy.

Norfloxacin, a frequently used ocular antibiotic, might have cytotoxic effect on human corneal endothelial cells (HCECs), subsequently damage the cornea and finally impair human vision. However, the possible mechanisms of cytotoxicity of norfloxacin to HCEC line are unclear. Herein, we investigated the cytotoxicity of norfloxacin and its underlying cellular and molecular mechanisms using in vitro cultured non-transfected HCECs and verified the cytotoxicity with cat corneal endothelium in vivo. In the present study, the cytotoxicity of norfloxacin in the in vitro cultured HCECs was recognized by causing abnormal morphology such as cell shrinkage and detachment from plate bottom, and decline of viability of in vitro cultured HCECs. Then, its cytotoxicity was verified by inducing reduction of cell density and morphological abnormality of in vivo cat corneal endothelial cells. Furthermore, the cytotoxicity of norfloxacin in HCECs was corroborated as apoptosis by elevation of plasma membrane permeability, S phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in in vitro cultured HCECs and apoptosis-like swollen cells in the in vivo model. Moreover, norfloxacin induced extrinsic death receptor-mediated apoptosis pathway by activating caspase-2/-8/-3 and intrinsic mitochondrion-dependent apoptosis pathway by downregulating anti-apoptotic Bcl-2 and upregulating of pro-apoptotic Bad, which disrupted mitochondrial transmembrane potential, subsequently upregulated cytoplasmic cytochrome c and apoptosis-inducing factor and finally activated caspase-9/-3. Generally, norfloxacin induces HCE cell apoptosis via a death receptor-mediated and mitochondrion-dependent signaling pathway.

Atropine, a widely used topical anticholinergic drug, might have adverse effects on human corneas in vivo. However, its cytotoxic effect on human corneal endothelium (HCE) and its possible mechanisms are unclear. Here, we investigated the cytotoxicity of atropine and its underlying cellular and molecular mechanisms using an in vitro model of HCE cells and verified the cytotoxicity using cat corneal endothelium (CCE) in vivo. Our results showed that atropine at concentrations above 0.3125 g/L could induce abnormal morphology and viability decline in a dose- and time-dependent manner in vitro. The cytotoxicity of atropine was proven by the induced density decrease and abnormality of morphology and ultrastructure of CCE cells in vivo. Meanwhile, atropine could also induce dose- and time-dependent elevation of plasma membrane permeability, G1 phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCE cells. Moreover, 2.5 g/L atropine could also induce caspase-2/-3/-9 activation, mitochondrial transmembrane potential disruption, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, upregulation of pro-apoptotic Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. In conclusion, atropine above 1/128 of its clinical therapeutic dosage has a dose- and time-dependent cytotoxicity to HCE cells in vitro which is confirmed by CCE cells in vivo, and its cytotoxicity is achieved by inducing HCE cell apoptosis via a death receptor-mediated mitochondrion-dependent signaling pathway. Our findings provide new insights into the cytotoxicity and apoptosis-inducing effect of atropine which should be used with great caution in eye clinic.

A novel continuous ovary cell line from barfin flounder (Verasper moseri) (BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22°C in Dulbecco’s modified Eagle medium/F12 medium (DMEM/F12, 1:1; pH 7.2) supplemented with 20% fetal bovine serum (FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22°C. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number (46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein (GFP) positively expressed in these cells after being transformed with pcDNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.

In the present study, the toxicity of phenylephrine, a selective α1-adrenergic receptor agonist, in corneal epithelial cells and its underlying mechanisms were investigated using an in vitro model of human corneal epithelial cells (HCEPCs) and an in vivo model of New Zealand white rabbit corneas. The HCEPCs treated with phenylephrine at concentrations from 10% to 0.078125% displayed abnormal morphology, decline of cell viability and elevation of plasma membrane permeability time- and dose-dependently. Moreover, 10%-1.25% phenylephrine induce necrosis characteristics of marginalization and uneven distribution of chromatin through up-regulation of RIPK1, RIPK3 and MLKL along with inactivation of caspase-8 and caspase-2, whereas 0.625% phenylephrine induced condensed chromatin, S phase arrest, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation in the HCECs through activation of caspase-2, -8, -9 and -3 as well as down-regulation of Bcl-2, up-regulation of Bad, ΔΨm disruption and release of cytochrome c and AIF into cytosol. At last, 10% phenylephrine induced destruction of the corneal epithelia and apoptosis of corneal epithelial cells in rabbit corneas. In conclusion, 10% to 1.25% phenylephrine cause necroptosis via RIPK1-RIPK3-MLKL axis and 0.625% phenylephrine induce apoptosis via a mitochondrion-dependent and death receptor-mediated signal pathway in HCEPCs.

Background Recently, many patients with corneal blindness caused by endothelial dysfunction have no opportunity to receive keratoplasty therapy because of the extremely limited number of donor corneas. Corneal tissue engineering opens a new path for in vitro reconstruction of tissue-engineered HCE which will cure the corneal endotheliopathy by clinical corneal transplantation. In this study, we construct a human corneal endothelium (HCE) equivalent with non-transfected monoclonal HCE (mcHCE) cells and modified denuded amniotic membrane (mdAM), and evaluate its functions in monkey models. Methods Tissue-engineered HCE (TE-HCE) was constructed by culturing DiI-labeled mcHCE cells on mdAMs in 20% fetal bovine serum-containing DMEM/Ham's Nutrient Mixture F12 (1:1) medium and 5% CO2 at 37°C on a 24-well culture plate. The constructed TE-HCE was transplanted into monkey corneas via penetrating keratoplasty with Descemet's membrane and endothelium stripped. The corneal transparency, thickness, and intraocular pressure were monitored in vivo, and the corneal morphology and histological structure were examined ex vivo 181 days after surgery. Results The constructed TE-HCE, with an average density of 3602.22 ± 45.22 cells/mm2 , mimicked its natural counterpart both in morphology and histological structure. In vivo, corneal transparency was maintained, and the corneal thickness gradually decreased to 567.33 ± 72.77 μm at day 181 after TE-HCE transplanted into monkey eyes, while intense corneal edema and turbid were found in mdAM-transplanted eyes with their corneal thicknesses maintained over 1000 μm during the monitoring period. Ex vivo, a monolayer of corneal endothelium, consisting of mcHCE cells at a density of 2795.65 ± 156.83 cells/mm2 , was reconstructed in transplanted monkey eyes. The cells in the transplanted area had the hexagonal or polygonal morphology and normal ultrastructure, and established plenty of cell-cell and cell-stromal matrix junctions. Besides, huge membrane-bounded flat stacks with electric dense inclusions were found in mcHCE cells beneath the plasma membrane at the stromal side. Conclusions The constructed TE-HCE has normal histological property and functions well in monkey models. The TE-HCE could be used as a promising HCE equivalent in therapy of corneal endothelium dysfunction and corneal regenerative medicine.

A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders.

Proparacaine is a widely used topical anesthetic in ophthalmic optometry and surgery, and has been reported to have cytotoxic effects on rabbit corneal endothelial cells after prolonged and repeated usage. Since rabbit is an exceptive mammal whose corneal endothelial cells still maintaining proliferation abilities even in adulthood, whether proparacaine has cytotoxic effects on human corneal endothelial (HCE) cells need to be further verified. Our objectives in the present study were to investigate the cytotoxicity to HCE cells of proparacaine and its underlying mechanisms in vitro and verify the cytotoxicity using cat corneal endothelial (CCE) cells in an in vivo model of cat corneas. Cytotoxic evaluation results indicated that a dose- and time-dependent toxic response of HCE cells to proparacaine over 0.03125% was rated based on morphology and viability, and a toxic response of CCE cells to 0.5% (clinical applied dosage) proparacaine was also rated based on cell density and histology. Importantly, treatment with proparacaine resulted in significant elevation of plasma membrane permeability, cell cycle arrest at S phase, fragmentation of genomic DNA, formation of apoptotic bodies, and externalization of phosphatidylserine (PS) of HCE cells. Moreover, proparacaine demonstrated disrupting effects on mitochondrial transmembrane potential (MTP) of HCE cells and activating effects on caspase-3, -8 and -9. This study demonstrates that proparacaine has notable cytotoxicity to both HCE cells in vitro and CCE cells in vivo, and its dose- and time-dependent cytotoxicity to HCE cells is achieved by inducing apoptosis via a mitochondrion-mediated caspase-dependent pathway. These findings provide new insights into the cytotoxicity and apoptosis-inducing effect of local anesthetics which should be used with great caution in the eye clinic.

Continuous cell culture of a puffer fish Takifugu rubripes has been established for efficient delivery of exogenous genes or proteins to cultured fish cells. Transcription factor oct4 was chosen for transduction into cultured fish cells because of its conserved structure and function between fish and mammals. In this work, the T. rubripes oct4 gene was cloned and expressed in Escherichia coli as a recombinant protein by introducing cell-penetrating peptide (CPP) poly-arginine (11R) and 6His-tag at the C-terminus. After purification, recombinant proteins were added to the growth medium and incubated with T. rubripes spermary cells. Recombinant proteins that crossed the cell membrane were detected in the cytoplasm and nucleus by western blot and immunofluorescent observation. The function of transduced oct4 as a transcription factor in fish cells was confirmed by driving green fluorescent protein expression in the pEGFP-1 reporter construct with the conserved specific oct4-binding sequence from mouse Mus musculus. Taken together, 11R can be an efficient CPP in delivering fusion proteins to cultured fish cells.

Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0 g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625 g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.

Lidocaine has been reported to induce apoptosis on rabbit corneal endothelial cells. However, the apoptotic effect and exact mechanism involved in cytotoxicity of lidocaine are not well-established in human corneal endothelial (HCE) cells. In this study, we investigated the apoptosis-inducing effect of lidocaine on HCE cells in vitro. After HCE cells were treated with lidocaine at concentrations of 0.15625-10.0 g/l, the morphology and ultrastructure of the cells were observed by inverted light microscope and transmission electron microscope (TEM). Cell viability was measured by MTT assay, and the apoptotic ratio was evaluated with flow cytometry and fluorescent microscopic counting after FITC-Annexin V/PI and AO/EB staining. DNA fragmentation was detected by electrophoresis, and the activation of caspases was evaluated by ELISA. In addition, changes in mitochondrial membrane potential were determined by JC-1 staining. Results suggest that lidocaine above 1.25 g/l reduced cellular viability and triggered apoptosis in HCE cells in a time- and dose-dependent manner. Diminishment of ΔΨm and the activation of caspases indicate that lidocaine-induced apoptosis was caspase dependent and may be related to mitochondrial pathway.

In this work, a sensitive analytical method to study the stability of two new series of synthesized heterocyclic compounds, the benzimidazole-4,7-diones 5 and N-oxide benzimidazole-4,7-dione derivatives 6 was established and validated. These derivatives were developed as potential anticancer substances to be activated under hypoxic conditions. At this point we were concerned with establishing their stability in some specific environments for further biological studies. For that, we developed and validated an RP-UPLC method. Next, selected compounds were tested in vitro for possible anticancer activity. Their effect on A549 tumour cell lines under normoxia and hypoxia conditions was determined by a WST-1 test. Four of the examined compounds (compounds 5a-c and 6c) showed very good antiproliferative effects and three of them (compounds 6a, 6b and 6d) were specific for hypoxia conditions. The hypoxia/normoxia cytotoxic coefficient of compound 6b is close to that of tirapazamine--a reference compound in our experiments--and this parameter locates it between mitomycin C and 2-nitroimidazole (misonidazole).

Purpose: To investigate the cytotoxic effect of latanoprost on corneal stroma and its underlying cellular and molecular mechanisms using non-transfected human corneal stromal (HCS) cells as an in vitro model. Methods: After HCS cells were treated with latanoprost at concentrations varying from 50 mg/l (clinical therapeutic dosage) to 0.78125 mg/l, and cell morphology, cell viability, and cell cycle were detected by light microscopy, methyl thiazolyl tetrazolium assay, and flow cytometry (FCM) with propidium iodide (PI) staining, respectively. Meanwhile, alterations in plasma membrane permeability, phosphatidylserine (PS) orientation, DNA integrality, and cell ultrastructure were examined by acridine orange (AO)/ethidium bromide (EB) double staining, FCM with Annexin-V/propidium iodide (PI) staining, DNA electrophoresis, and transmission electron microscopy. Furthermore, caspase activation, mitochondrial transmembrane potential (MTP), and expression of pro-apoptotic regulators were determined by ELISA, FCM with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethybenzimida (JC-1) staining, and Western blot, respectively. Results: Latanoprost above concentrations of 3.125 mg/l can induce dose- and time-dependent morphological abnormality, growth retardation, viability decline, and plasma membrane permeability elevation of HCS cells. Moreover, latanoprost can arrest the cell cycle of these cells at S phase and induce PS externalization, DNA fragmentation, and apoptotic body formation of the cells. Furthermore, latanoprost can induce activation of caspase-3, -8 and -9; disruption of MTP; downregulation of anti-apoptotic Bcl-2; upregulation of pro-apoptotic Bax; and cytoplasmic cytochrome c release. Conclusions: Latanoprost above and at 3.125 mg/l (1/16 of its clinical therapeutic dosage) has a dose- and time-dependent cytotoxicity to HCS cells by inducing death receptor-mediated mitochondria-dependent apoptosis, which should be used with great caution in clinical situations to avoid undesired damages to HCS cells.

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy.

Bivalve hemoglobins have antibacterial activities, while the underlying mechanisms remain poorly understood. In our study, three full-length cDNAs of hemoglobins from blood clam skHbs were obtained, encoding putative polypeptides of 147, 150, and 152 amino acids, respectively. Predicted advanced protein structures showed that the skHbs had amphipathic antibacterial structures, displayed the typical structural characteristics of proteins with globin-like fold containing numerous alpha-helixes, and forming a homodimeric skHbI and a heterotetrameric skHbII complex. After injected with alive and heat-killed Gram-positive bacteria Bacillus subtilis, the mRNA levels of skHbI and skHbII were both significantly upregulated through increasing the expression of peptidoglycan recognition protein-like (PGRP-like) protein and Toll-like receptor (TLR-like) protein induced by peptidoglycan on the surface of the bacteria, but there were no obvious differences in their protein levels. Besides, reactive oxygen species (ROS) was detected to participate in the resistance to B. subtilis. These implied that skHbs could involve in the innate immune responses to Gram-positive bacterial infection directly with their amphipathic structures and indirectly by increasing ROS production through PGRP triggering Toll pathway. In conclusion, our findings reveal the structural characteristics of skHbs and their mechanism against Gram-positive bacteria thereby providing the molecular evidence for fundamental innate antibacterial activities by invoking respiratory proteins.

Phenylephrine (PHE), a selective α1-adrenergic receptor agonist, is often used as a decongestant for mydriasis prior to cataract surgery, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of PHE to the cornea and its cellular and molecular mechanisms remain unknown. To provide references for secure medication and prospective therapeutic interventions of PHE, we investigated the cytotoxicity of PHE to corneal stroma and its possible mechanisms using an in vitro model of human corneal stromal (HCS) cells and an in vivo model of cat keratocytes. We found that PHE, above the concentration of 0.0781125% (1/128 of its clinical therapeutic dosage), had a dose- and time-dependent cytotoxicity to HCS cells by inducing morphological abnormality and viability decline, as well as S phase arrest. Moreover, PHE induced apoptosis of HCS cells by inducing plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation. Furthermore, PHE could induce activations of caspase-3 and -9, disruption of mitochondrial transmembrane potential, downregulation of anti-apoptotic Bcl-xL, upregulation of pro-apoptotic Bax, along with upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. The cytotoxic and pro-apoptotic effects of PHE were also proven by the induced apoptotic-like ultrastructural alterations of keratocytes in vivo. Taken together, our results suggest that PHE has a significant cytotoxicity to corneal stroma cells both in vitro and in vivo by inducing cell apoptosis, and the pro-apoptotic effect of PHE is achieved via a Bcl-2 family proteins-mediated mitochondrion-dependent pathway.

AIM: To demonstrate the cytotoxic effect and possible mechanisms of Tetracaine on human corneal epithelial (HCEP) cells in vitro. METHODS: In vitro cultured HCEP cell were treated with Tetracaine hydrochloride at different doses for different times, and their morphology, viability, and plasma membrane permeability were detected by light microscopy, methyl thiazolyl tetrazolium (MTT) assay, and acridine orange (AO)/ethidium bromide (EB) staining, respectively. Their cell cycle progression, phosphatidylserine orientation in plasma membrane, and mitochondrial membrane potential (MTP) were assessed by flow cytometry. DNA fragmentation, ultrastructure, caspase activation, and the cytoplasmic apoptosis inducing factor (AIF) and cytochrome c (Cyt. c) along with the expression of B-cell lymphoma-2 (Bcl-2) family proteins were examined by gel electrophoresis, transmission electron microscope, enzyme linked immunosorbent assay (ELISA), and Western blot, respectively. RESULTS: After exposed to Tetracaine at doses from 10.0 to 0.3125 g/L, the HCEP cells showed dose- and time-dependent morphological abnormality and typical cytopathic effect, viability decline, and plasma membrane permeability elevation. Tetracaine induced phosphatidylserine externalization, DNA fragmentation, G1 phase arrest, and ultrastructural abnormality and apoptotic body formation. Furthermore, Tetracaine at a dose of 0.3125 g/L also induced caspase-3, -9 and -8 activation, MTP disruption, up-regulation of the cytoplasmic amount of Cyt. c and AIF, the expressions of Bax and Bad, and down-regulation of the expressions of Bcl-2 and Bcl-xL. CONCLUSION: Tetracaine above 0.3125 g/L (1/32 of its clinical applied dosage) has a dose- and time-dependent cytotoxicity to HCEP cells in vitro, with inducing cell apoptosis via a death receptor-mediated mitochondrion-dependent pathway.

Human corneal endothelium (HCE) is vital in maintaining corneal transparency and thickness, and damages to it may result in endotheliopathy and even corneal blindness, which can only be cured by keratoplasty. Due to the shortage of donor corneas, tissue-engineered HCE (TE-HCE) has become an equivalent to HCE. This study constructs a high endothelial cell density (ECD) TE-HCE and evaluates its functions by corneal transplantation in rabbits. A TE-HCE was constructed by culturing DiI-labeled nontransfected HCE cells on modified denuded amniotic membrane (mdAM) using collagen IV, fibronectin, and laminin, and rabbit corneas were characterized both in vivo and ex vivo after TE-HCE transplantation. Our results indicated that the constructed TE-HCE with a high ECD of 3611 ± 56.66 cells/mm2 had similar morphology and structure to a native HCE. In vivo postsurgery detections showed that the TE-HCE-transplanted cornea gained transparency and recovered its thickness gradually during a monitoring period of 358 days, while the mdAM-transplanted cornea remained opaque and edematous. Ex vivo examinations revealed that a native-like corneal endothelium with an ECD of 2703 ± 70.37 cells/mm2 was reconstructed by the transplanted TE-HCE. Our findings suggested that the TE-HCE might be used as a HCE equivalent and has promising applications in therapy of corneal endotheliopathy.

Turbot (Scophthalmus maximus L.) reddish body iridovirus (TRBIV) was propagated in turbot fin cells (TF cells) and inactivated as the TRBIV vaccine with its protection efficiency evaluated in this study. TF cells were cultured in 10% bovine calf serum (BCS)-containing MEM medium (pH7.0) at 22°C, in which TRBIV propagated to a titer as high as 105.6 TCID50 mL−1. The TRBIV was inactivated with 0.1% formalin and formulated with 0.5% aluminum hydroxide. The inactivated vaccine caused neither cytopathogenic effect (CPE) on TF cells nor pathogenic effect on turbots. After being administered with the vaccine twice via muscle injection, the turbot developed high-tittered TRBIV neutralizing antibodies in a dose-dependent manner. The vaccine protected the turbot from dying with an immunoprotection rate of 83.3% as was determined via subcutaneous vaccination in the laboratory and 90.5% via bath vaccination in turbot farms, respectively. The inactivated vaccine was very immunogenic, efficiently preventing turbot from death. It holds the potential of being applied in aquaculture.

To understand the defense mechanisms of Crustacean animals, brine shrimp Artemia sinica prophenoloxidase (AsproPO) cDNA was cloned and its expression at early developmental stages was examined by reverse-transcription PCR (RT-PCR) and semi-quantitative RT-PCR, and activity of phenoloxidase (PO) at different developmental stages was further detected by using l-3,4-dihydroxyphenylalanine (l-DOPA) as a specific substrate in this study. It was found that the full-length of AsproPO cDNA is 2125 bp and it contains an open reading frame of 2100 bp encoding a protein of 699 amino acids. The deduced amino acid sequence of AsproPO has two putative copper binding sites highly conserved in Arthropods. Semi-quantitative RT-PCR analyses showed that the gene of AsproPO expressed at Emergence, Instar I and Instar II stages but did not at 0 h and 6 h stages. Activity measurement showed that PO activity could only be detected at Instar II stage but the other measured stages. All these implied that Artemia proPO immune system was complexly modulated during early development.

A large yellow croaker, Pseudosciaena crocea, spleen (LYCS) cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study. Primary culture of LYCS cells was initiated from spleen tissue pieces, which were cultured at 25°C in Dulbecco’s modiced Eagle medium/F12 medium (DMEM/F12, 1:1) (pH7.2), supplemented with 20% fetal bovine serum, carboxymethyl chitosan, chondroitin sulfate, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The cultured LYCS cells, in fibroblast shape, proliferated to 100% confluency 20 days later. Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P. crocea (6m+6sm+36t, NF=60). A LYCS cell line, with a population doubling time of 48.7 h at passage 60, has been established and subcultured to passage 70. Transgenic feasibility test demonstrated that positive green fluorescence protein (GFP) expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection. In conclusion, a continuous foreign gene transfection feasible LYCS cell line has been established successfully. The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.

To understand the mechanisms of starfish regeneration, the arms of adult starfish Asterias rollestoni Bell were amputated and their regeneration patterns and cellular mechanisms were studied. It was found that cells in the outer epidermis and inner parietal peritoneum near the end of the stump began to dedifferentiate 4 d after amputation. The dedifferentiated cells in the outer epidermis proliferated, migrated to the wound site and formed a thickened pre-epidermis which would then re-differentiate gradually into mature epidermis. The new parietal peritoneum formed on the coelomic side of wound might be from the curvely elongated parietal peritoneum, resulting from the dedifferentiated and proliferated cells by extension. Afterwards, the proliferated cells made the outer epidermis and inner parietal peritoneum invaginate into the interior dermis and formed blastema-like structures together with induced dedifferentiated dermal cells. Most interestingly, the arm regeneration in A. rollestoni was achieved synchronously by de novo arm-bud formation and growth, and arm-stump elongation. The crucial aspects of arm-bud formation included cell dedifferentiation, proliferation and migration, while those of arm-stump elongation included cell dedifferentiation, proliferation, invagination, and arm-wall-across blastema-like structure formation. The unique pattern and cellular mechanisms of amputated arm regeneration make it easier to understand the rapid regeneration process of adult starfish. This study may lay solid foundations for the research into molecular mechanisms of echinoderm regeneration.

To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP), TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mcHCECs) and modified denuded amniotic membranes (mdAMs) were transplanted into PCEP models of New Zealand white rabbits using penetrating keratoplasty. The TE-HCEs were examined using diverse techniques including slit-lamp biomicroscopy observation and pachymeter and tonometer measurements in vivo, and fluorescent microscopy, alizarin red staining, paraffin sectioning, scanning and transmission electron microscopy observations in vitro. The corneas of transplanted eyes maintained transparency for as long as 200 d without obvious edema or immune rejection. The corneal thickness of transplanted eyes decreased gradually after transplanting, reaching almost the thickness of normal eyes after 156 d, while the TE-HCE non-transplanted eyes were turbid and showed obvious corneal edema. The polygonal corneal endothelial cells in the transplanted area originated from the TE-HCE transplant. An intact monolayer corneal endothelium had been reconstructed with the morphology, cell density and structure similar to those of normal rabbit corneal endothelium. In conclusion, the transplanted TE-HCE can reconstruct the integrality of corneal endothelium and restore corneal transparency and thickness in PCEP rabbits. The TE-HCE functions normally as an endothelial barrier and pump and promises to be an equivalent of HCE for clinical therapy of human PCEP.

Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China. The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitable for assessing genetic diversity. Four microsatellite-enriched libraries of half smooth tongue sole (Cynoglossus semilaevis) were constructed, from which 57 polymorphic microsatellites were isolated and characterized. The polymorphism of these microsatellites was assessed by genotyping in 30 individual fish. The number of alleles ranged from 2 to 11, with an average of 4.614 alleles per locus. The values of observed and expected heterozygosities ranged from 0.1000 to 1.0000 and from 0.0966 to 0.8847 respectively. Polymorphism information content (PIC) ranged from 0.0905 to 0.862. These markers would be useful for population structure assessment, genetic linkage map construction and parentage analysis for this species.

Cell growth in primary cell culture of the brine shrimp (Artemia sinica) embryo at 12 and 20 h after rehydration at 25°C was examined comparatively in modified Leibovitz-15 medium. The cells from A. sinica embryo at 12 h after rehydration were dispersed, and the cells disseminated but did not attach to the surface of wells and multiply at 2 d of culture, and 12 d later, the cells were degenerated and dead. The best growth of the brine shrimp cells was obtained from the prenauplii of A. sinica at 20 h after dormant embryo rehydration. The fibroblast-like cells attached to the well surface and multiplied at 15 d after the primary culture was set up. Confluent monolayer was formed at 50 d. The prenauplii cells have been subcultured up to passage 3 and maintained for approximately 200 d. The reasons for cell growth potential at the different developmental stages of Artemia embryo were discussed.

A cell line, SHK, was derived from the kidney of spotted halibut Verasper variegates. The cell line was subcultured more than 40 passages in minimum essential medium (MEM) supplemented with fetal bovine serum (FBS) and 10 ng ml−1 basic fibroblast growth factor (bFGF). Cell morphology from primary culture and subculture was observed continuously by microscopy. The SHK cell line consisted predominantly of fibroblast-like cells. The cell line was able to grow between 20°C and 30°C with the optimum growth at 24°C and with a reduced growth between 12°C and 20°C. The growth rate of the cells increased as the proportion of FBS increased from 10% to 20% at 28°C with optimum growth at the concentration of 20%. The doubling time of the cells was determined to be 44.8 h. Chromosome analysis revealed that 52% of the SHK cells maintained a normal diploid chromosome number (2n=46). The cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies. The cells were infected by lymphosystis disease virus (LCDV) and found to be susceptible to the virus in cytopathic effect (CPE) observation. The infection was confirmed by PCR and electron microscopy experiments, which proved the existence of the viral particles in the cytoplasm of the virus-infected cells.

A novel cell line (bmGH) was established from the heart of brown-marbled grouper Epinephelus fuscoguttatus and its viral susceptibility was evaluated. The bmGH cells have been subcultured to passage 65 in Dulbecco's modified eagle medium:Ham's nutrient mixture F-12 (1:1) medium (DMEM/F12) which was further supplemented with foetal bovine serum (FBS), carboxymethyl-chitosan, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) at 24 degrees C. The heart cells have a fibroblastic morphology and proliferated to confluence 14 days later. The cells grew at a steady rate during subsequent subculture and had a population doubling time of 40.3 h at passage 60. Karyotype analysis showed that these cells exhibited chromosomal aneuploidy with a modal chromosome number of 48. The results of viral susceptibility characterization revealed that cytopathic effects (CPE) of bmGH cells appeared after infection by two iridoviruses, turbot reddish body iridovirus (TRBIV) and lymphocystis disease virus (LCDV). A large number of TRBIV and LCDV particles were also observed in the infected bmGH cells by electron microscope examination. All of these facts indicate that the bmGH cell line established here may serve as a valuable tool for studies of cell-virus interactions and has potential applications in fish virus isolation, propagation and vaccine development.

By using Artemia chorion as a specific substrate, the hatching enzyme from Artemia salina (AHE) was purified by gel-filtration and ion-exchange chromatography, and characterized biochemically and enzymatically in this study. It was found that the AHE had a molecular weight of 82.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and often contained 73.3 kDa molecules in preparation. The AHE had obvious choriolytic activity, which was optimal at pH 7.0 and a temperature of 40°C. The K m value of the AHE for dimethyl casein was 8.20 mg/ml. The AHE activity was almost completely inhibited by soybean trypsin inhibitor and p-amidinophenyl methane sulfonyl fluoride hydrochloride, greatly inhibited by N-tosyl-l-lysyl chloromethyl ketone, phenylmethanesulfonyl fluoride, and lima bean trypsin inhibitor, slightly inhibited by pepstatin, N-tosyl-l-phenylalanyl chloromethyl ketone, leupeptin, N-ethylmaleimide, and iodoacetamide, and not inhibited by chymostatin and bestatin. All these results imply that AHE is most probably a trypsin-type serine protease. Besides of these, AHE was also sensitive to EDTA and Zn 2+ . Combined with the results that the EDTA-pre-treated HE activity could be perfectly recovered by Zn 2+ , it is indicated that AHE might be also a kind of Zn-metalloprotease.

Phenoloxidase (PO) from ink sacs of Octopus ocellatus was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its biochemical and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. It was found that prophenoloxidase from O. ocellatus was isolated as a heterodimeric protein of 153.8 kDa, and two subunits of 75.6 and 73.0 kDa were often detected in preparations after SDS activation. The PO-like activity showed optimal pH of 7.0, optimal temperature of 40 degrees C, and an apparent Km value of 3.1 mM on L-DOPA, and 6.3 mM on catechol, respectively. The PO-like activity was extremely sensitive to 1-phenyl-2-thiourea and sodium sulfite, and very sensitive to ascorbic acid, thiourea, citric acid, and benzoic acid. Together with its specific enzyme activity on catechol and L-DOPA, it can be concluded that the Octopus PO is most probably a typical o-diphenoloxidase. The PO-like activity was also strongly inhibited by Cu(2+), Zn(2+), ethylenediaminetetraacetic acid and diethyldithiocarbamate (DETC), and the DETC-inhibited PO-like activity could be perfectly restored by Cu(2+). These results indicated that Octopus PO is most probably a copper-containing metalloenzyme. All these results implied that the PO from O. ocellatus has the properties of a catechol-type copper-containing o-diphenoloxidase which functions not only as a catalytic enzyme in melanin production in ink sacs but also as a humoral factor in host defense via melaninization as in other crustaceans.

To lay a solid foundation of in vitro investigations of fish viral diseases, cytotechnology and cytotoxicology, a novel fin cell line from brown-marbled grouper, Epinephelus fuscoguttatus, was established and its viral susceptibility was evaluated. The fin tissues, digested with hyaluronidase and collagenase II, were used to initiate primary culture at 24 °C by using 20% foetal bovine serum-Dulbecco's modified Eagle medium/F12 medium, which was further supplemented with carboxymethyl–chitooligosaccharide, basic fibroblast growth factor and insulin-like growth factor-I. The fibroblastic fin cells grew at a steady rate during subsequent subculture and had a population doubling time of 50.6 h at passage 60. The modal diploid chromosome number was 48. A brown-marbled grouper fin cell line (bmGF-1) has been established and subcultured to passage 75 by now. Viral susceptibilities revealed that typical cytopathic effects of bmGF-1 cells emerged after being infected by turbot reddish-body iridovirus (TRBIV) or lymphocystis disease virus (LCDV). However, a large number of TRBIV and LCDV particles were also found in infected bmGF-1 cells. All these indicate that the bmGF-1 cell line has good susceptibility to TRBIV and LCDV, which may serve as a valuable tool for studies of cell–virus interactions and have potential applications in fish virus propagation and vaccine development.

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immunostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.

To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37 degrees C, 5% CO(2). The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

Proparacaine is a widely used topical anesthetic in ophthalmic optometry and surgery, and has been reported to have cytotoxic effects on rabbit corneal endothelial cells after prolonged and repeated usage. Since rabbit is an exceptive mammal whose corneal endothelial cells still maintaining proliferation abilities even in adulthood, whether proparacaine has cytotoxic effects on human corneal endothelial (HCE) cells need to be further verified. Our objectives in the present study were to investigate the cytotoxicity to HCE cells of proparacaine and its underlying mechanisms in vitro and verify the cytotoxicity using cat corneal endothelial (CCE) cells in an in vivo model of cat corneas. Cytotoxic evaluation results indicated that a dose- and time-dependent toxic response of HCE cells to proparacaine over 0.03125% was rated based on morphology and viability, and a toxic response of CCE cells to 0.5% (clinical applied dosage) proparacaine was also rated based on cell density and histology. Importantly, treatment with proparacaine resulted in significant elevation of plasma membrane permeability, cell cycle arrest at S phase, fragmentation of genomic DNA, formation of apoptotic bodies, and externalization of phosphatidylserine (PS) of HCE cells. Moreover, proparacaine demonstrated disrupting effects on mitochondrial transmembrane potential (MTP) of HCE cells and activating effects on caspase-3, -8 and -9. This study demonstrates that proparacaine has notable cytotoxicity to both HCE cells in vitro and CCE cells in vivo, and its dose- and time-dependent cytotoxicity to HCE cells is achieved by inducing apoptosis via a mitochondrion-mediated caspase-dependent pathway. These findings provide new insights into the cytotoxicity and apoptosis-inducing effect of local anesthetics which should be used with great caution in the eye clinic.

AIM: To examine the cytotoxic effect of pilocarpine, an anti-glaucoma drug, on human corneal stromal (HCS) cells and its underlying cytotoxic mechanisms using an in vitro model of non-transfected HCS cells. METHODS: After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L, their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability, DNA fragmentation and ultrastructure were examined by acridine orange (AO)/ethidium bromide (EB) double-staining. DNA electrophoresis and transmission electron microscopy (TEM), cell cycle, phosphatidylserine (PS) orientation and mitochondrial transmembrane potential (MTP) were assayed by flow cytometry (FCM). And the activation of caspases was checked by ELISA. RESULTS: Morphology observations and viability assay showed that pilocarpine at concentrations above 0.625 g/L induced dose- and time-dependent morphological abnormality and viability decline of HCS cells. AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or time-dependent membrane permeability elevation, DNA fragmentation, and apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and caspase-8, -9 and -3 activation of the cells. CONCLUSION: Pilocarpine at concentrations above 0.625 g/L (1/32 of its clinical therapeutic dosage) has a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in these cells, which is most probably regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.

By using Penaeus chorion as a specific substrate, the hatching enzyme (HE) from Penaeus chinensis was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular weight of Penaeus HE is about 43.0 kDa in SDS–PAGE. The Penaeus HE had obvious choriolytic activity, which was optimal at pH 6.0 and temperature of 40 °C, respectively. The Km value of the HE for casein was 7.47 mg ml−1. The HE activity was almost completely inhibited by SBTI, p-APMSF, bestatin, and NEM, greatly inhibited by ovomucoid, TLCK, IAM, chymostatin, and PMSF, and slightly inhibited by pepstatin A, TPCK, LBTI, and leupeptin. These results indicate that the HE is most probably a trypsin-type serine protease. Besides of these, the HE was extremely sensitive to EDTA, Zn2+, Ca2+, Mg2+, and Cu2+. Combined with the results that the EDTA-pretreated HE activity could be perfectly recovered by Zn2+, it is indicated that shrimp HE is most probably a kind of Zn-metalloprotease.

The evolutionary race between hosts and pathogens has led to a variety of adaptations. Little is known about the immunological role of hemoglobin (Hb) in antimicrobial immune responses. Results showed that a 31.2 kDa monodimer Hb (skHbI) and a 57.8 kDa heterotetramer Hb (skHbII) from the blood clam, Scapharca kagoshimensis, had phenoloxidase (PO)-like activities and antimicrobial activities. Both were found capable of oxidizing l-DOPA, catechol and hydroquinone. Their PO-like activities were visibly greatly inhibited by oxidase inhibitors, EDTA, and divalent metal ions, and greatly enhanced by isopropanol and Fe(2+), indicating that they have the properties of a metalloenzyme and a catecholase-type PO as well. They also showed obvious anti-bacterial activities against gram-positive bacteria but not against either gram-negative bacteria nor fungi. The anti-bacterial activities levels were a result of the generation of reactive oxygen species (ROS) of superoxide anions. These results indicate that skHbI and skHbII, not only function as iron-containing oxygen carriers, but also exert anti-bacterial activities and catecholase-type oxidizing activities. The fact that skHbII exerts high level of PO-like activity indicates different roles in the innate immunodefense system. These results may improve understanding of the multiple functions of invertebrate Hbs beyond serving as oxygen carriers and may provide insight into how the fundamental and universal mode of the innate immune system has persisted in respiratory proteins throughout the course of evolution.

Using L-dihydroxyphenylalanine (L-DOPA) as a specific substrate, phenoloxidase (PO) from clam (Ruditapes philippinarum) was purified by Q Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-100 gel-filtration, and characterized biochemically and enzymatically in this study. The molecular mass of PO in SDS-PAGE is about 76.9 kDa, and the prophenoloxidase (proPO) molecule, isolated as a monomeric protein, is 84.1 kDa. The PO molecule had a high oxidative activity, and the proPO molecule had almost no oxidative activity. The PO activity was optimal at pH 7.0 and temperature of 40 degrees C. The Km value of the PO for L-DOPA was 2.2 mmol l(-1). The PO was extremely sensitive to benzoic acid and sodium sulfite, very sensitive to citric acid, thio urea, 1-phenyl-2-thiourea and cysteine, but not sensitive to ascorbic acid. Combined with its specific enzyme activity on tyrosine and L-DOPA, it can be concluded that the Ruditapes PO is probably a kind of tyrosinase-type phenoloxidase. The PO activity was strongly inhibited by ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DETC), Zn2+, Ca2+ and Cu2+, as well as by Mg2+. The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu2+ on DETC-inhibited PO activity, indicate that Ruditapes PO is most probably a copper-containing metalloenzyme.

Lymphocystis disease virus (LCDV), a large icosahedral DNA virus classified to the iridovirus family, is the causative agent of lymphocystis, a disease which occurs in marine and freshwater fish species and is characterized by formation of papilloma-like lesions on the surface of the skin. In vitro, LCDV infection causes flounder gill cells, an adherent cell line, to exhibit an obvious cytopathic effect (CPE). In order to test whether apoptosis is responsible for the observed CPE, cells infected with LCDV at a multiplicity of infection (m.o.i.) of 5 PFU per cell were examined at various time intervals for the appearance of apoptotic signs. Nuclear fragmentation, DNA laddering and caspase activation were observed in the infected cells at the time (i.e. 10 days post-infection) when an intensive CPE was observed. These findings demonstrate that LCDV is capable of inducing apoptosis in vitro, which is different from the result of LCDV infection in vivo, and consequently suggest an intricate LCDV-host interaction.

Purpose To evaluate the performance of reconstructed tissue-engineered human corneal endothelium (TE-HCE) by corneal transplantation in cat models. Methods TE-HCE reconstruction was performed by culturing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled monoclonal HCE cells on denuded amniotic membranes (dAMs) in 20% fetal bovine serum-containing Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F12 (1:1) medium and 5% CO2 at 37 °C on a 24-well culture plate. The reconstructed TE-HCE was transplanted into cat corneas via lamellar keratoplasty with all of the endothelium and part of Descemet’s membrane stripped. Postsurgical corneas were monitored daily with their histological properties examined during a period of 104 days after transplantation. Results The reconstructed TE-HCE at a density of 3,413.33±111.23 cells/mm2 in average established intense cell-cell and cell-dAM junctions. After lamellar keratoplasty surgery, no obvious edema was found in TE-HCE-transplanted cat corneas, which were transparent throughout the monitoring period. In contrast, intense corneal edema developed in dAM-transplanted cat corneas, which were turbid. The corneal thickness gradually decreased to 751.33±11.37 μm on day 104 after TE-HCE transplantation, while that of dAM eye was over 1,000 μm in thickness during the monitoring period. A monolayer of endothelium consisting of TE-HCE-originated cells at a density of 2,573.33±0.59 cells/mm2 attached tightly to the surface of remnant Descemet’s membrane over 104 days; this was similar to the normal eye control in cell density. Conclusions The reconstructed TE-HCE was able to function as a corneal endothelium equivalent and restore corneal function in cat models.

Phenoloxidase from Artemia sinica (AsPO) was purified by Superdex 200 gel-filtration and Q Sepharose fast flow ion-exchange chromatography, and its properties were characterized biochemically and enzymatically by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. Results showed that AsPO was isolated as a monomeric protein of 125.5 kDa in molecular mass. The optimal pH value and temperature are 7.0 and 50°C, respectively, for its PO activity. The AsPO had an apparent K(m) value of 4.2 mM on L-DOPA, and 10.9 mM on catechol, respectively. Oxidase inhibitor on PO activity showed that the AsPO was extremely sensitive to ascorbic acid, sodium sulfite, and citric acid; and was very sensitive to cysteine, benzoic acid, and 1-phenyl-2-thiourea. Combined with its specific enzyme activity on L-DOPA and catechol, it can be concluded that AsPO is most probably a typical catechol-type O-diphenoloxidase. Its PO activity was also sensitive to metal ions and chelators, and 20 mM DETC-inhibited PO activity was obviously recovered by 15 mM Cu(2+), indicating that AsPO is most probably a copper-containing metalloenzyme. All these data about specific substrate, sensitivity to oxidase inhibitor metal ions and chelators indicate that the AsPO has the properties of a catechol-type copper-containing O-diphenoloxidase that functions as a vital humoral factor in host defense via melaninization as in other Crustaceans.