The impact of oleanolic and ursolic acid on corneal epithelial cells in vitro

INTRODUCTION. Oleanolic (OA) and ursolic (UA) acids belong to the triterpene group widely present in plants. These compounds are recognised to have anti-inflammatory properties and thus are considered to be used in therapies as well as in cosmetic, natural health, or diet products. AIM. The scientific hypothesis of our study was to show that OA and UA influence corneal epithelial cells cultured in vitro. METHODS. Toxicity tests, based on MTT and Neutral Red (NR) uptake, measurement of nitric oxide (NOx) level, as well as analysis of metalloproteinases (MMP-2 and MMP-9) amount and activity were performed. RESULTS . UA expressed significantly higher toxicity on cells than OA. At the lowest concentration applied (5 μM), UA limited cellular metabolism and viability on average by 22% as compared to untreated control, while 25 μM resulted in values lower than 10%. On the other hand, OA at the highest (100 μM) concentration limited cellular metabolism and viability by about 20%. NOx level significantly increased when OA and UA were applied at concentrations of 25 and 100 μM, respectively. OA and UA had a stronger impact on the level of MMP-2 than MMP-9. OA and UA reduced MMP-2 and MMP-9 in the whole range of concentrations. Tested triterpenoids had no significant impact on MMP activity. CONCLUSIONS. OA and UA have a different impact on human corneal epithelial cells. UA is toxic for corneal epithelial cells, while OA exhibits milder activity, which may be useful for further analysis in ocular pharmacology

Reactivity of corneal and conjunctival epithelial cells to lipopolysaccharide (LPS) and/or irradiation with visible light in vitro

INTRODUCTION. Visible light and inflammation caused by bacterial endotoxins strongly influence direct cell interactions and modulate the expression of selected factors, such as nitric oxide (NO) and cyclooxygenase-2 (COX-2). The aim of the study is to establish whether exposition of corneal or conjunctival epithelial cells to visible light and/ or LPS may change their viability, direct cellular interactions and expression of NO and COX-2. MATERIALS AND METHODS. In vitro cultured human corneal and conjunctival epithelial cells were used in the study. The following assays were performed: Neutral Red (NR) uptake, nitric oxide (NO) quantification by the Griess method, cytoskeletal F-actin organization by fluorescent staining, and COX-2 expression by immunofluorescence. RESULTS. LPS reduced the viability of the cells, especially conjunctival epithelial cells. All cell stimulation variants tested (visible light and/or LPS treatment) led to decreased nitric oxide (NO) production both by corneal and conjunctival epithelial cells. No changes in cytoskeletal F-actin filaments were observed after the cells had been treated with light or the endotoxin. LPS slightly increased COX-2 expression, but light had no, or a slightly reducing, effect on the level of this enzyme. CONCLUSIONS. Visible light and/or bacterial endotoxin (LPS) may, depending on the local microenvironmental conditions, cooperate or interfere with each other’s activity in inducing ocular surface inflammation

The role of collagen in co-cultures of human normal corneal and conjunctival cells

Background: Intermediate interactions between corneal and conjunctival epithelial cells play an important rolein the process of correct vision. The goal of this paper was to establish whether the presence or absence of collagentype I changes paracrine interactions between corneal and conjunctival epithelial cells. Material and methods: Cultures of human corneal and conjunctival epithelial cells were used in the study. TheELISA quantitative analysis of interleukin 1β (IL-1β), interleukin 6 (IL-6), urokinase-type plasminogen activator(uPA), and uPA receptor (uPAR), assessment of the type of interactions between cells, as well as correlations betweentested parameters were performed. Results: The presence of collagen type I changed the quantitative production of IL-1β and IL-6 by the examinedcells in the co-culture system. It did not affect the level of released uPA and uPAR. The presence or absence ofcollagen also changed the relationship between the cells, which were evaluated in relation to changes in the level ofreleased cytokines. Conclusions: Different levels of collagen type I constituting a component of extracellular matrix proteins significantlyaffect and regulate the indirect interactions between human corneal and conjunctival epithelial cells

Homocysteine influences the human keratocytes cell cycle

BACKGROUND: Homocysteine (Hcy), a metabolic intermediate, is a sulfur-containing amino acid not present in the structure of proteins. It has been shown that high Hcy levels via oxidative stress, induced inflammation, and vessel dysfunction may affect the functioning of tissues, including the structures that form the eye, among others keratocytes. The visual disturbance caused by high levels of Hcy may also be associated with disturbed cell proliferation resulting from the effect of this amino acid on the cell cycle. The goal was to analyse the influence of Hcy on the keratocytes and to find out in which phase of the cell cycle its course is disturbed by this amino acid. MATERIALS AND METHODS: A normal human keratocytes (HK) cell line was used in the study. May-Grünwald-Giemsa (MGG) staining for morphology visualization and cytometric cell cycle analysis were performed. RESULTS: Hcy does not affect the G1 phase of the cycle, while it regulates the S and G2 phases. Changes in the amount of the sub-G1 population  indicative of a pro-apoptotic effect of Hcy on keratocytes were detected. The form of the Hcy administered (L stereoisomer or DL racemic mixture) and the amino acid concentration were also important. CONCLUSIONS: Homocysteine influences the keratocyte cell cycle. The change occurs at the stage of the G1-S transition, which suggests a decreasing level of cells in the S phase and an increasing level in the G2 phase. The long-term influence of Hcy on keratocytes may affect keratocytes proliferation and possible cornea regeneration

Signals in solid-state photochemically induced dynamic nuclear polarization recover faster than signals obtained with the longitudinal relaxation time

Biological and Soft Matter PhysicsSolid state NMR/Biophysical Organic Chemistr

Nanosecond-Flash N-15 Photo-CIDNP MAS NMR on reaction centers of Rhodobacter sphaeroides R26

Solid state NMR/Biophysical Organic ChemistryBiological and Soft Matter Physic

Observation of the solid-state photo-CIDNP effect in entire cells of cyanobacteria Synechocystis

Cyanobacteria are widely used as model organism of oxygenic photosynthesis due to being the simplest photosynthetic organisms containing both photosystem I and II (PSI and PSII). Photochemically induced dynamic nuclear polarization (photo-CIDNP) 13C magic-angle spinning (MAS) NMR is a powerful tool in understanding the photosynthesis machinery down to atomic level. Combined with selective isotope enrichment this technique has now opened the door to study primary charge separation in whole living cells. Here, we present the first photo-CIDNP observed in whole cells of the cyanobacterium Synechocystis

Magnetic field dependence of photo-CIDNP MAS NMR on photosynthetic reaction centers of Rhodobacter sphaeroides WT

Biological and Soft Matter Physic

Photo-CIDNP MAS NMR in intact cells of Rhodobacter sphaeroides R26: molecular and atomic resolution at nanomolar concentration?

Biological and Soft Matter Physic