Normal Human Lenses Research Articles

Dielectric response to thermal denaturation of lenses in healthy and diabetic rabbits

The relative permittivity and conductivity of healthy and alloxane-induced diabetic rabbits lenses were measured over a frequency range of 500 Hz to 100 kHz in an electric field and at temperatures from 25 to 150 °C. The dielectric spectra for both tissues showed two separate relaxations with a characteristic frequency of around 4 and 25 kHz assigned to the cortical and nuclear zones, respectively. These two dispersions are due to the interfacial polarization at the surface of the α-crystallin molecules. The denaturation temperature for the non-diabetic lens and the diabetic lens is approximately 70 and 80 °C, respectively. Moreover, the relative permittivity and conductivity values are higher in the diabetic lens than in the non-diabetic tissue at the same temperature and frequency. Our dielectric studies provide a better understanding of the thermal stability of crystallin-water complexes in normal and diseased human lenses.

Aging-dependent loss of GAP junction proteins Cx46 and Cx50 in the fiber cells of human and mouse lenses accounts for the diminished coupling conductance.

The homeostasis of the ocular lens is maintained by a microcirculation system propagated through gap junction channels. It is well established that the intercellular communications of the lens become deteriorative during aging. However, the molecular basis for this change in human lenses has not been well defined. Here, we present evidence to show that over 90% of Cx46 and Cx50 are lost in the fiber cells of normal human lenses aged 50 and above. From transparent to cataractous lenses, while Cx43 was upregulated, both Cx46 and Cx50 were significantly down-regulated in the lens epithelia. During aging of mouse lenses, Cx43 remained unchanged, but both Cx46 and Cx50 were significantly downregulated. Under oxidative stress treatment, mouse lenses develop in vitro cataractogenesis. Associated with this process, Cx43 was significantly upregulated, in contrast, Cx46 and Cx50 were sharply downregulated. Together, our results for the first time reveal that downregulation in Cx46 and Cx50 levels appears to be the major reason for the diminished coupling conductance, and the aging-dependent loss of Cx46 and Cx50 promotes senile cataractogenesis.

Upregulation of miR-630 Induced by Oxidative Damage Resists Cell Migration Through Targeting ALCAM in Human Lens Epithelium Cells

ABSTRACTPurpose: To investigate the role of miRNAs in regulating oxidative damage during cataract formation.Methods: Microarray analysis and gene expression profiling assay were used to separately evaluate the miRNAs and mRNAs profiles in normal human lens epithelium cell line HLE-B3 treated by H2O2. The expression level of miR-630 was detected by RT-qPCR and the gene expression profiles were performed with gene ontology analysis using Bio Informatical database. The targets of miR-630 were predicted using miRecords and the results were used for screening targets of miR-630 combined with the GO analysis above. The mRNA levels of ALCAM, PCDH7, COL12A2, and EDIL3 in HLE-B3 cells after oxidative stimulation or miR-630 mimics transfection were measured by RT-qPCR, and the expression of ALCAM regulated by miR-630 was confirmed by Western blot and dual-luciferase reporter gene assay. The level of cell migration was measured by transwell assay and scratching test after transfection of miR-630 mimics and ALCAM siRNAs.Results: The microarray analysis demonstrated that miR-630 was significantly increased in HLE-B3 cells after oxidative stimulation. ALCAM, PCDH7, COL12A2, and EDIL3 were screened to be the possible targets of miR-630 by miRecords combined with GO analysis, but the results of RT-qPCR, Western blot and dual-luciferase reporter gene assay showed that only the expression of ALCAM was repressed by miR-630 transfection. Cell migration was inhibited through transfection of miR-630 mimics or ALCAM siRNAs and the upregulation of miR-630 partly reduced the cell migration increased by oxidative stimulation.Conclusion: miR-630 is one of the miRNAs increased by oxidative stimulation in human lens epithelium cells. Its upregulation may inhibit cell migration by targeting on ALCAM, which is important for HLECs to resist behavioral changes induced by oxidative damage and may delay the progression of cataract.

Study on iTRAQ-based differential expression protein analysis in nuclear cataract lens with different axial length

Objective To identify and verify differentially expressed proteins in lens of nuclear cataract eyes with different axial lengths. Methods Forty-eight nuclear cataract patients with the hardness grade Ⅲ to Ⅳ were enrolled from January 2016 to December 2017 in the east area of Xining city.The lens nucleus was obtained during extracapsular cataract extraction (ECCE) surgery.The samples were divided into six groups according to age and axial length, with 8 samples in each group.Seven normal transparent human lens nuclei were obtained from donors during corneal transplantation surgery and served as normal control.Water-soluble, water-insoluble and water-insoluble-urea-soluble protein fractions were extracted separately and merged to analyze by using 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with 2D-LC/MS/MS.The data were processed with ProteinPilot software.Two technical replicates of iTRAQ analyses were carried out.Up-regulated and down-regulated proteins were selected in two replicates as candidate biomarkers.Twenty lens nuclei from 20 nulear cataract patients were divided into long axis young age group, normal axis young age group, long axis old age group and normal axis old age group.Western blot was used to confirm the most different proteins.This study protocol was approved by the Ethics Committee of TangDu Hospital of Air Force Medical University (TDLL2015046). Written consent was obtained from each participant. Results Eighty proteins were identified.Six proteins were selected as differentially-expressed proteins.Sorbitol dehydrogenase (SDH) was elevated whereas beta-crystallin B2 (CRYBB2) was decreased in the long axis groups.The relative expression of SDH protein in the long axis low age group was 1.204±0.142, which was significantly higher than 0.711±0.072 in the normal axis low age group, the relative expression level of SDH in the long axis old age group was 1.092±0.334, which was significantly higher than 0.617±0.089 in the normal axis old age group, the difference was statistically significant (all at P<0.05). Conclusions The expression levels of SDH in the nuclear cataract were elevated with long axial length.SDH may be involved in the pathogenesis of axial myopia-associated nuclear cataract. Key words: Axial lengths, eye; Human lens, crystalline; Nuclear cataract; Isotope labeling; Proteomics

MicroRNA-378a Regulates the Reactive Oxygen Species (ROS)/Phosphatidylinositol 3-Kinases (PI3K)/AKT Signaling Pathway in Human Lens Epithelial Cells and Cataract.

BackgroundCataract is associated with increased apoptosis of the epithelial cells of the ocular lens. Previous studies have shown that microRNA-378a (miR-378a) has a role in the development of cataract, but the molecular mechanisms remain unclear. This study aimed to investigate the effects of miR-378a in human lens epithelial cells (HLECs) in vitro and normal lens tissues and cataract tissues.Material/MethodsHLECs were grown in culture. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot were used to examine gene expression levels. The MTT and TUNEL assay measured cell growth and apoptosis. Changes in the fluorescence ratio of ethidium to dihydroethidium (E: DHE) and in 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (C-H2DCFDA) were used to detect superoxide (O2−) and hydrogen peroxide (H2O2). The expression levels of miR-378a and the superoxide dismutase 1 gene (SOD1) were measured in normal human lens tissues and cataract tissues.ResultsUpregulation of miR-378a reduced the expression of SOD1. Levels of O2− were upregulated and H2O2 was slightly down-regulated by miR-378a. The use of a miR-378a mimic suppressed cell growth and enhanced apoptosis of HLECs, which were reversed by the use of a miR-378a inhibitor. SOD1 overexpression rescued the miR-378a-induced phenotypes of HLEC cells. Treatment with the PI3K inhibitor, LY294002, reversed miR-378a and ROS-regulated proliferation and apoptosis of HLEC cells. Also, miR-378a was upregulated, and SOD1 was down-regulated in human cataract tissues.ConclusionsIn HLECs, expression of miR-378a regulated ROS and PI3K/AKT signaling, and miR-378a was upregulated, and SOD1 was down-regulated in human cataract tissue.

Age-related spatial differences of human lens UV filters revealed by negative ion mode MALDI imaging mass spectrometry

Tryptophan-derived UV filters are predominantly found in the lenses of primates and humans. While protective against UV radiation, aging alters the complement and spatial distributions of human lens UV filters, and a role for UV filters has been suggested in age-related cataract formation. To establish how the spatial distributions of UV filters change in normal human lens aging, matrix assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) was utilised to map the locations and relative abundance of multiple UV filters simultaneously. Frozen human lenses were cryosectioned axially, and the 20 μm-thick sections coated with MALDI matrix via robotic sprayer and analysed using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS. While signal for many UV filters was detected throughout the lenses, signal intensity was generally highest in the central (embryonic) nucleus and decreased uniformly in outer (foetal, juvenile, adult) nuclear and cortical regions, and many UV filter signals declined with age. In contrast, two antioxidant-conjugated UV filters (Cys-3-OHKG and GSH-3-OHKG) were restricted to the lens nucleus and their relative signal increased with increasing lens age. The enhanced spatial resolution of MALDI-IMS over manual trephine dissection techniques and its multiplex capability allowed the spatial relationships between lens UV filters to be established and explored in relation to aging. Together these results confirmed that the complement of UV filters in each lens is dynamic and undergoes significant age-related changes. In the future, this information could be used to compare with other lens biomolecule changes to better understand the lens aging process and age-related cataract formation.

Novel production method of innovative antiangiogenic and antitumor small peptides in Escherichia coli.

BackgroundDeveloping innovative drugs with potent efficacy, specificity, and high safety remains an ongoing task in antitumor therapy development. In the last few years, peptide drugs have become attractive agents in cancer therapy. HM-3, mainly with antiangiogenic effect, and AP25, with an additional antiproliferative effect, are two peptides designed in our laboratory targeting αvβ3 and α5β1 integrins, respectively. The low molecular weight of the two peptides renders their recombinant expression very difficult, and the complicated structure of AP25 makes its chemical synthesis restricted, which presents a big challenge for its development.MethodsBifunctional peptides designed by the ligation of HM-3 and AP25, using linkers with different flexibility, were prepared using recombinant DNA technology in Escherichia coli. The fusion peptides were expressed in a modified auto-induction medium based on a mixture of glucose, glycerol, and lactose as carbon substrates and NH4+ as nitrogen source without any amino acid or other elements. Subsequently, the antiangiogenic, antiproliferative, and cell adhesion assays were conducted to evaluate the bioactivity of the two fusion peptides.ResultsThe peptides were successfully expressed in a soluble form without any induction, which allows the culture to reach higher cell density before protein expression occurs. Human umbilical vein endothelial cell migration assay and chick embryo chorioallantoic membrane assay showed, at low doses, a significantly increased antiangiogenic effect (>75%) of the purified products compared with the single molecules. Meanwhile, MTT assay confirmed their enhanced antitumor activity against gastric cancer cell line MGC-803; however, no significant effect was observed on hepatoma HepG2 cells and no cytotoxicity on normal human lens epithelial cell SRA01/04 and human epithelial esophageal cells.ConclusionBifunctional molecules with antiangiogenic and antiproliferative effects were obtained by using this technique, which presents an alternative for small peptide production, instead of the conventional chemical method. The increased molecular weight facilitates the peptide expression with a simultaneous improvement in their stability and biological activity.

Racemization at the Asp 58 residue in αA-crystallin from the lens of high myopic cataract patients.

Post‐translational modifications in lens proteins are key causal factors in cataract. As the most abundant post‐translational modification in the lens, racemization may be closely related to the pathogenesis of cataract. Racemization of αA‐crystallin, a crucial structural and heat shock protein in the human lens, could significantly influence its structure and function. In previous studies, elevated racemization from l‐Asp 58 to d‐isoAsp58 in αA‐crystallin has been found in age‐related cataract (ARC) lenses compared to normal aged human lenses. However, the role of racemization in high myopic cataract (HMC), which is characterized by an early onset of nuclear cataract, remains unknown. In the current study, apparently different from ARC, significantly increased racemization from l‐Asp 58 to d‐Asp 58 in αA‐crystallin was identified in HMC lenses. The average racemization rates for each Asp isoform were calculated in ARC and HMC group. In ARC patients, the conversion of l‐Asp 58 to d‐isoAsp 58, up to 31.89%, accounted for the main proportion in racemization, which was in accordance with the previous studies. However, in HMC lenses, the conversion of l‐Asp 58 to d‐Asp 58, as high as 35.44%, accounted for the largest proportion of racemization in αA‐crystallin. The different trend in the conversion of αA‐crystallin by racemization, especially the elevated level of d‐Asp 58 in HMC lenses, might prompt early cataractogenesis and a possible explanation of distinct phenotypes of cataract in HMC.

Ionizing radiation response of primary normal human lens epithelial cells.

Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.

Long noncoding RNA expression profile in HLE B-3 cells during TGF-\u03b22-induced epithelial-mesenchymal transition

BackgroundRecent evidence has shown that long noncoding RNAs (lncRNAs) are involved in the process of epithelial-mesenchymal transition (EMT). However, little research has focused on the expression profile of lncRNAs during EMT in human lens epithelial cells (LECs) and their functions have not yet been described.MethodsDysregulated lncRNAs and mRNAs in normal human lens epithelial B-3(HLE B-3) cells and during transforming growth factor β2(TGF-β2)-induced EMT were analyzed via lncRNA microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analyses of differentially expressed mRNAs were performed to identify their functions and pathologic pathways. Six candidate lncRNAs were validated via quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) to confirm the microarray data.ResultsA total of 775 lncRNAs (325 up-regulated and 450 down-regulated) and 935 mRNAs (329 up-regulated and 606 down-regulated) were differentially expressed in HLE B-3 cells during TGF-β2-induced EMT compared to normal HLE B-3 cells. GO and KEGG Pathway analyses indicated the functions of differentially expressed mRNAs in the TGF-β2-induced EMT in HLE B-3 cells. qRT-PCR confirmed the trends indicated in microarray analysis for all 6 candidate lncRNAs.ConclusionOur study lays the foundation for future research in lncRNAs related to EMT in HLE B-3 cells and could provide new avenues for the prevention and treatment of posterior capsule opacification (PCO).

Plumbagin Suppresses α-MSH-Induced Melanogenesis in B16F10 Mouse Melanoma Cells by Inhibiting Tyrosinase Activity.

Recent studies have shown that plumbagin has anti-inflammatory, anti-allergic, antibacterial, and anti-cancer activities; however, it has not yet been shown whether plumbagin suppresses alpha-melanocyte stimulating hormone (α-MSH)-induced melanin synthesis to prevent hyperpigmentation. In this study, we demonstrated that plumbagin significantly suppresses α-MSH-stimulated melanin synthesis in B16F10 mouse melanoma cells. To understand the inhibitory mechanism of plumbagin on melanin synthesis, we performed cellular or cell-free tyrosinase activity assays and analyzed melanogenesis-related gene expression. We demonstrated that plumbagin directly suppresses tyrosinase activity independent of the transcriptional machinery associated with melanogenesis, which includes micropthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP1). We also investigated whether plumbagin was toxic to normal human keratinocytes (HaCaT) and lens epithelial cells (B3) that may be injured by using skin-care cosmetics. Surprisingly, lower plumbagin concentrations (0.5–1 μM) effectively inhibited melanin synthesis and tyrosinase activity but do not cause toxicity in keratinocytes, lens epithelial cells, and B16F10 mouse melanoma cells, suggesting that plumbagin is safe for dermal application. Taken together, these results suggest that the inhibitory effect of plumbagin to pigmentation may make it an acceptable and safe component for use in skin-care cosmetic formulations used for skin whitening.

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2.

To study the inhibition of nuclear factor kappa-B p65 (NF-κB p65) antisense oligodeoxynucleotide (ASODN) on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2 (TGF-β2) in vitro. NF-κB p65 ASODN and NF-κB p65 missense oligodeoxynucleotide (MSODN) were designed and synthesized. Human lens epithelial cell line (HLE B-3) cells were prepared for study and divided into 7 groups. Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium (DMEM). T1, T2, and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/mL TGF-β2 for 6h, 12h, 24h respectively. A+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 ASODN for 24h. M+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h. The negative control group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after cultured with transfer agent (HiPerFect) for 24h. Cell morphology was observed at different time points using an inverted microscope. The expression of NF-κB p65 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the expression of α-smooth muscle actin (α-SMA) protein was assayed with ELISA. With the TGF-β2 stimulation prolongation, the expression of NF-κB p65 mRNA and α-SMA protein increased in T1, T2, T3 groups compared with the control group, and the difference was statistically significant (P<0.05). NF-κB p65 ASODN lowered the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. NF-κB p65 MSODN and HiPerFect did not lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. The difference between control group and A+T group was not statistically significant (P>0.05), but the difference among A+T group and other groups was statistically significant (P<0.05). NF-κB p65 ASODN could lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2, and antagonized TGF-β2-induced transdifferentiation of HLE B-3 in vitro. NF-κB p65 ASODN could be used as a new biological therapeutic target of posterior capsular opacification.

Spatial distribution of metabolites in the human lens

Spatial distribution of 34 metabolites along the optical and equatorial axes of the human lens has been determined. For the majority of metabolites, the homogeneous distribution has been observed. That suggests that the rate of the metabolite transformation in the lens is low due to the general metabolic passivity of the lens fiber cells. However, the redox processes are active in the lens; as a result, some metabolites, including antioxidants, demonstrate the “nucleus-depleted” type of distribution, whereas secondary UV filters show the “nucleus-enriched” type. The metabolite concentrations at the lens poles and equator are similar for all metabolites under study. The concentric pattern of the “nucleus-depleted” and “nucleus-enriched” distributions testifies that the metabolite distribution inside the lens is mostly governed by a passive diffusion, relatively free along the fiber cells and retarded in the radial direction across the cells. No significant difference in the metabolite distribution between the normal and cataractous human lenses was found.

Cytomorphometric study of epithelial cells in normal and cataractous human lenses in relation with hyperglycemia.

The aim of the study is to evaluate and correlate the morphology and cell density of epithelial cells adhering to lens capsule surgically removed from the anterior central region with lens clarity and type of cataract present in patients with or without type 2 diabetes. Capsulorhexis specimens were obtained from patients who had undergone phacoemulsification cataract surgery. All the samples were centrifuged and stained by the aid of Papanicolaou technique and were observed under light microscope. We determinated the mean cell density, the degree of epithelial damage, and morphological indicators of cells such as cell area and the nucleus-plasma ratio. Patients with cataract demonstrated a statistical significant decrease in cell density and an heterogeneous cell picture in which enlarged cells dominated. In addition, type 2 diabetics with cataract had a significantly even lower mean epithelial cell density by the presence of larger cell area with smaller nucleus-plasma ratio. More pronounced alterations in the lens epithelium were correlated not only with the presence of cortical cataract, increased fasting blood sugar, and increased HbA1c but also with the prolonged duration of diabetes and the co-existence of diabetic retinopathy. It seems that density and morphology of the anterior lens epithelial cells determine the lens epithelium damage which is more profound in hyperglycemia and in cortical cataracts. The changes in lens epithelium seem to play an important role in cataractogenesis.

Quantitative proteomics analysis by iTRAQ in human nuclear cataracts of different ages and normal lens nuclei.

The goal of this study was to quantitatively identify the differentially expressed proteins in nuclear cataracts of different ages and normal lens nuclei in humans. Forty-eight human lens nucleus samples with hardness grades III, IV were obtained during cataract surgery by extracapsular cataract extraction. Seven normal transparent human lens nuclei were obtained from fresh normal cadaver eyes during corneal transplantation surgery. Lens nuclei were divided into seven groups according to age and optic axis: Group A (average age 80.8 ± 1.2 years), Group B (average age 57.0 ± 4.0 years), Group C average age 80.3 ± 4.5 years), Group D (average age 56.9 ± 4.2 years), Group E (average age 78.1 ± 2.5 years), Group F (average age 57.6 ± 3.3 years) and Group G (seven normal transparent human lenses from normal cadaver eyes, average age 34.7 ± 4.2 years). Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were further analyzed using 8-plex iTRAQ labeling combined with 2D-LC-MS/MS. The data were analyzed with the ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated by Western blotting. We employed biological and technical replicates and selected the intersection of the two results, which included 80 proteins. Nine proteins were differentially expressed among the 80 proteins identified using proteomic techniques. In age-related nuclear cataracts (ARNC), the expression levels of fatty acid-binding protein and pterin-4-alpha-carbinolamine dehydratase were upregulated, whereas the levels of alpha-crystallin B chain (CRYAB), GSH synthetase, phakinin, gamma-crystallin C, phosphoglycerate kinase 1, betaine-homocysteine S-methyltransferase 1 (BHMT1), and spectrin beta chain were downregulated. These proteins may be associated with abnormal protein aggregation and oxidative stress. GSH synthetase and CRYAB expression levels in the nuclear cataract decreased with age. The mass spectrometric analysis results were consistent with the Western blot validation. The results indicate that CRYAB and GSH synthetase may be involved in ARNC pathogenesis. iTRAQ combined with 2D-LC-MS/MS provides new methods for future studies of pathological mechanisms and protective drug development for ARNC.

Metabolomic composition of normal aged and cataractous human lenses

Quantitative metabolomic profiles of normal and cataractous human lenses were obtained with the combined use of high-frequency nuclear magnetic resonance (NMR) and high-performance liquid chromatography with high-resolution mass-spectrometric detection (LC-MS) methods. The concentration of more than fifty metabolites in the lens cortex and nucleus has been determined. For the majority of metabolites, their concentrations in the lens cortex and nucleus are similar, which confirms low metabolic activity in the lens core. The difference between the metabolite levels in the cortex and nucleus of the normal lens is observed for antioxidants and UV filters, which demonstrates the activity of redox processes in the lens. A huge difference is found between the metabolomic compositions of normal and age-matched cataractous lenses: the concentrations of almost all metabolites in the normal lens are higher than in the cataractous one. The most pronounced difference is observed for compounds playing a key role in the lens cell protection and metabolic activity, including antioxidants, UV filters, and osmolytes. The results obtained imply that the development of the age-related cataracts might originate from the metabolic dysfunction of the lens epithelial cells.

Efficient production of native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein system in Escherichia coli.

To develop an efficient and cost-effective approach for the production of small preventive peptide lunasin with correct natural N terminus, a synthetic gene was designed by OPTIMIZER & Gene Designer and cloned into pTWIN1 vector at SapI and PstI sites. Thus, lunasin was N-terminally fused to the pH-induced self-cleavable Ssp DnaB mini-intein linked to a chitin binding domain (CBD) with no extra residues. The resultant fusion protein was highly expressed by lactose induction in Escherichia coli BL21 (DE3) in a 7-l bioreactor and bound to a chitin affinity column. After washing the impurities, the Ssp DnaB intein mediated on-column self-cleavage was easily triggered by shifting pH and temperature to allow the native lunasin released. The final purified lunasin yielded up to 75 mg/l medium. Tricine/SDS-PAGE and matrix-assisted laser desorption time-of-flight (MALDI-TOF)/mass spectrometry (MS) verified the structural authenticity of the product, implying the correct cleavage at the junction between Ssp DnaB intein and lunasin. MTT assay confirmed its potent proliferation inhibitory activity to human cancer cells HCT-116 and MDA-MB-231; however, no cytotoxicity to normal human lens epithelial cell SRA01/04 and hepatoma HepG2. Taken together, we provide a novel strategy to produce recombinant native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein.

Loss of calpastatin leads to activation of calpain in human lens epithelial cells.

Activation of calpains (calpain 2 and Lp82) in rodent lenses readily causes proteolysis and cataract formation. In contrast, primate lenses are quite resistant to activation of calpains. The hypothesis is that high levels of human endogenous calpain inhibitor, calpastatin (CS), prevent calpain activation in human lenses. The purpose of the present study was to directly test if CS is a major inhibitory factor in a human lens epithelial cell line, HLE B-3. Small interfering RNAs (siRNAs) were used to knock down expression of CS in HLE B-3. The cells then were cultured with the calcium ionophore, ionomycin, with or without a calpain inhibitor SNJ-1945. Transcripts for calpain 2 and CS were measured by quantitative PCR (qPCR). Calpain 2 activity was detected by immunoblotting for the calpain-specific, α-spectrin breakdown product and for activation-associated, fragments of calpain 2. Expression of CS in HLE B-3 was remarkably higher than in α-TN4 (mouse comparator cell line). Proteolysis of α-spectrin was observed in the soluble proteins from α-TN4 incubated with Ca(2+), but not in the human HLE B-3. When CS-reduced HLE B-3 cells (transfected with CS siRNA) were cultured with ionomycin, calpain 2 was activated, specific proteolysis of α-spectrin occurred, and cell death ensued; SNJ-1945 inhibited these changes. Our data demonstrated that the high levels of endogenous CS do, indeed, inhibit calpain activity in normal human lens epithelial cells. We speculate that age-related oxidation might cause loss of CS activity in human lens epithelial cells, allowing activation of long-dormant calpain 2, proteolysis of critical cytoskeletal proteins, and cataract formation.

Presence and distribution of l-kynurenine aminotransferases immunoreactivity in human cataractous lenses

To investigate the presence and distribution of l-kynurenine aminotransferases immunoreactivity in human and animal lenses during cataract formation. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, KAT II and KAT III on sections of 26 anterior capsules from patients undergoing surgical treatment of anterior subcapsular cataract (ASC) and 22 cataractous lenses from human eyes enucleated because of choroidal malignant melanoma. Additionally, the eyes of 11-month-old DBA/2J mice (6 eyes) were investigated (with KAT I and II). Ten clear human lenses and four BL6 mice lenses were used as controls. Spatial immunoreactivity patterns of enzymes were compared with Periodic Acid - Schiff (PAS)-stained sections. Immunohistochemical analysis revealed presence of KAT I, KAT II and KAT III in extracellular structures of all studied types of cataract in human eyes showing specific pattern of the stain. In cortical cataract, immunoreactivity was observed on cortical lens fibres. In nuclear cataract, KAT II revealed stronger and diffused staining than KAT I. Additionally, both KAT showed more pronounced staining at the edge of small clefts. In normal human lenses, KAT I, II and III, immunoreactivity was not observed. Presence of KAT I and KAT II in the intercellular substance of DBA/2J mice cataract was observed. In BL6 mice lenses without cataract, only weak KAT I and KAT II staining was observed. Presence of l-kynurenine aminotransferases in extracellular matrix (ECM) during human cataract formation suggests that products of l-kynurenine pathway might be involved in mechanisms of cataractogenesis.

Accelerated aging of Asp 58 in αA crystallin and human cataract formation

Racemisation of amino acids is one of the most abundant modifications in long-lived proteins. In this study racemisation of Asp 58 in the small heat shock protein, αA crystallin, was investigated. In normal human lenses, levels of l-isoAsp, d-isoAsp and d-Asp increased with age, such that by age 70 they accounted for approximately half of the total Asp at this site. Levels of d-isoAsp were significantly higher in all cataract lenses than age-matched normal lenses. The introduction of d-isoAsp in αA crystallin could therefore be associated with the development of cataract. Its more rapid formation in cataract lenses may represent an example of accelerated protein aging leading to a human age-related disease.