Effect of Disulfide Bond Incorporation on the Structure and Activity of Endostatin Peptide.

The structure and function of a 27-a.a. fragment of the N-terminal sequence of human endostatin (ES-Zn) were compared to those of the mutant peptide (ES-SSZn) obtained by adding Cys-Pro-Ala to the endostatin N-terminus and substituting Asn16 for Cys ensuring formation of a disulfide bond. Structural comparison of ES-Zn and ES-SSZn by far-UV circular dichroism (CD), intrinsic fluorescence, and molecular dynamics simulation methods revealed significant structural perturbations in ES-SSZn, such as elimination of the ß-sheet conformer, modification of the N-terminal loop structure, and reorganization of dynamic properties of the entire peptide backbone. ES-SSZn was approximately 2 and 3 times less efficient than ES-Zn and the full-length human endostatin, respectively, in the induction of caspase-3-dependent apoptosis in human umbilical vein endothelial cells (HUVECs) in vitro (p < 0.05). In contrast, treatment of metastatic 4T1 breast tumors in mice with ES-Zn and ES-SSZn (5 mg/kg body weight daily) for 14 days resulted in similar regression of tumor size, comparable downregulation of angiogenesis (CD31 and CD34) and cell proliferation (Ki67), and therefore, the same extent of apoptosis induction (TUNEL, p53, and Bcl-2) for both peptides (as compared to the untreated controls). Western blot analysis of HUVEC and 4T1 tumor lysates revealed the same levels of suppression of key signaling mediators Akt and ERK1/2 by ES-Zn and ES-SSZn. Contrary to the earlier studies, our results showed that the function of the 1-27 endostatin fragment is independent of its overall structure. Stabilization of the N-terminal loop structure by the disulfide bond incorporation causes relief from structural deviations.

Nonenzymatic antioxidants in saliva of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody-directed self-antigens, immune complex formation and immune deregulation, resulting in damage to essentially all the organs. SLE is associated with the increased production of free radicals. Increase in free radicals or impaired antioxidant defense system in SLE causes oxidative stress. Considering that saliva could be a reflection of the state of health, the purpose of this study was to evaluate some antioxidants in the saliva and serum of patients with SLE and compare these with healthy individuals. This could help us in obtaining a possible marker in saliva in the future. During the course of the practical part of the project, 30 patients with SLE and 30 healthy controls were investigated. After centrifugation of un-stimulated saliva and blood samples, they were examined using spectrophotometric methods and the results were analyzed by statistical software. According to the results, concentrations of malondialdehyde, uric acid and total antioxidants were significantly increased but the level of reduced glutathion was reduced significantly in the saliva and serum of SLE patients as compared to controls. It is therefore suggested that antioxidant power is impaired in saliva and serum of SLE patients. As there was a positive correlation between the antioxidant level of saliva and blood serum, the antioxidant status of saliva could be an indicator of serum antioxidants.

Alternations of salivary antioxidant enzymes in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic systemic inflammation. Oxidative stress may play a role in the pathogenesis of SLE. An increase in free radicals or an impaired antioxidant defense system in SLE causes oxidative stress. Therefore, oxidative damage plays an important role in the pathogenesis of SLE. Variations in antioxidant activity have been previously studied in serum of patients with this disease. However, salivary factors have not been evaluated. Considering that saliva, the noninvasive biological fluid, could be a reflection of the state of health, the purpose of this study was evaluation of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity in the saliva of patients with SLE. During the course of the practical part of the project, 30 patients with SLE and 30 healthy controls were selected to donate their saliva samples. After centrifugation of un-stimulated saliva, biological activity of POD, CAT and SOD were evaluated on their appropriate substrates using spectrophotometric methods and the results were statistically analyzed. The results showed that activities of antioxidant enzymes SOD and CAT were significantly reduced in saliva of SLE patients as compared to controls. The results suggest that antioxidant status was impaired in the saliva of SLE patients, and antioxidant status of saliva could be one of the non-invasive markers for SLE.

Tear proteins in health, disease, and contact lens wear.

Although well known as manifestations of sorrow, emotions, frustration, and blackmail, tears have a more prosaic and important function as a lubricant and as a blood substitute for the cornea. Tears transport oxygen and carbon dioxide and play a central role in the cellular economy of the ocular surface and conjunctiva. In addition to proteins, tears contain lipids and glycoproteins, which increase the wetting effect of the aqueous component and delay evaporation. The total protein concentration of tears is about 10% of that of the plasma. About 80 proteins and polypeptide components have been detected by electrophoresis. Among 30 proteins identified in tears, about 50% are enzymes. Some of the tear enzymes are secreted by the lacrimal glands; others are produced by or released from epithelial cells of the cornea and the conjunctiva. Finally, a few enzymes originate from plasma and appear in tears only in cases with increased permeability of the conjunctival vessels. The aim of this review is to provide clinical and biochemical information about tear enzymes both for ophthalmologists and for biochemists interested in clinical and experimental tear enzymology.

Variations in electrophoretic tear protein pattern due to contact lens wear.

Electrophoresis of tear proteins was used to compare the patterns from tear proteins in contact lens wearers with non-contact lens wearers. Thirty-five non-contact lens wearers and 35 wearers volunteered to enter our study. Both groups aged 20-25 years with no history of eye diseases. Stimulated tear samples were collected using a capillary tube. Total tear protein was measured for each group individually using standard assay methods. The results showed slightly higher total tear protein in the group wearing contact lenses. Some new bands also appeared in the electrophoresis patterns of this group as compared to the control group.

Biopolyesters from microorganisms: Biochemical basis of microbial synthesis, properties and applications.

Biopolyester produced by microorganisms has many useful properties for commercial and environmental applications. Poly(3-hydroxyalkonates) [poly(HA)s] and poly (â -malate) are among the best known biopolyesters produced by microor-ganisms. Poly(HA)s having a wide variety of repeating units can be produced by bacteria and they all posses very high biodegradability and biocompatibility. Poly (â -malate) is especially of interest for biomedical applications including drug delivery systems. During the last 20 yrs a large volume of research work has focused on the physiology, biochemistry and applications of poly(HA)s. In this review, the biochemical basis of bacterial production, the biodegradation properties and applications of poly(HA)s are considered.

Effect of poly-HEMA hydrophilic gel environment on the photo-physical behavior of rhodamine dyes.

The absorption spectra of rhodamine B (RB) chloride, rhodamine 6G (R6G) tetrafluoroborate and rhodamine 6G chloride in poly-2-hydroxyethyl methacrylate hydrogel (PHEMA) matrix were studied using absorption spectroscopy in the visible region. The transport and aggregative properties of these ionic dyes in aqueous solution across the hydrophilic gel were also investigated. The similarities of absorption spectra of RB in aqueous solutions and in hydrogel host suggest that the hydrogel framework has a minor effect in their absorption spectra. In contrast, there is a relatively strong interaction or electrostatic forces between R6G dyes and the hydrogel matrix. The permeability of R6G chloride through hydrogel host is seen to be markedly higher than RB chloride and R6G tetrafluoroborate.

Protein interaction with hydrogel contact lenses.

The first event observed at the interface between a contact lens and tear fluid is protein adsorption. Tears have a rich and complex composition, allowing a wide range of interactions and competitive processes. The majority of studies avail-able on protein adsorption have focused on blood/plasma/serum protein on solid surfaces. However, recently the interaction of tear proteins with contact lenses has become an important field of research following the widespread use of contact lenses in many physiological and pathological conditions. Protein adsorption on contact lenses is the overall result of various types of in-teractions between the different components present, i.e. the chemical composition and the surface charge, the structure of the protein molecules, the nature of the medium (tears) and many other solutes present in tears. The chemical structure of contact lenses and their physical and biological behavior are considered, followed by tear film structure and biochemistry. Finally, the nature and mechanism of protein adsorption on contact lenses, factors affecting adsorption and attempts to reduce protein ad-sorption are reviewed. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 1-19).

Competitive adsorption of proteins on hydrogel contact lenses.

PURPOSE: To compare sequential and competitive adsorption of some proteins onto hydrogel contact lenses. METHODS: New, never-worn, group IV soft contact lenses were incubated in freshly prepared solutions of lysozyme, alpha-lactalbumin, and ribonuclease at room temperature for 5 days with constant shaking, during which time their adsorbed protein was directly measured using UV absorbances at 280 nm. Adsorption isotherms were prepared. The lenses were rinsed with distilled water following a second protein treatment. RESULTS: We found that the chemical composition of the lens and the charge of the previously adsorbed protein affected sequential and competitive adsorption of proteins on a contact lens surface. CONCLUSIONS: Sequential adsorption occurs only if the second protein has a more favorable electrostatic interaction; sequential adsorption involves almost total displacement of the preadsorbed protein.

Aggregate formation of Rhodamine 6G in anisotropic solvents.

The aggregation of Rhodamine 6G in liquid crystalline solutions was studied using polarised spectroscopy and in a guest-host system based on homogeneous-homeotropic alignment. The orientation of the dye molecules (guest) was controlled using an electric field, and this enabled the contrast ratio R of the dye to be obtained by electrically switching. The excitonic treatment of the absorption spectra suggests a coplanar structure for the dimeric aggregates.

Lipid and protein deposition of N-vinyl pyrrolidone-containing group II and group IV frequent replacement contact lenses.

PURPOSE: The purpose of this study was to investigate the degree of protein and lipid deposition that occurs on N-vinyl-2-pyrrolidone (NVP) containing group II (non-ionic, high water content) and NVP containing group IV (ionic, high water content) frequent replacement hydrogel contact lens materials. METHODS: Twenty subjects were fitted with Group II (Vasurfilcon A) and Group IV (Vifilcon A) contact lenses, which were replaced monthly. The lenses were worn as a contralateral pair for 3 consecutive monthly periods. At the end of each monthly period, the lenses were collected for analysis of protein and lipid deposits. Protein deposition (following extraction) was examined by transmission UV and lipoidal deposition was examined using fluorescence spectrophotofluorimetry. RESULTS: There was a significant difference in the lipid and protein deposition profiles between the two materials. The Group II lens deposited approximately 2x more lipid (38 versus 73 fluorescence units; P < 0.0001) and the Group IV lens deposited approximately 17x more protein (488 micrograms versus 28 micrograms; P < 0.0001). Whilst the mean results across months were not significantly different for either protein or lipid (P = NS), the results revealed significant inter- and intra-subject variation. CONCLUSIONS: Protein deposition was predominantly controlled by the ionic charge of the lens materials, whereas the lipid deposition was predominantly determined by the NVP content. This study demonstrates that inter-subject variation and material characteristics significantly influence the deposition profile of hydrogel contact lens materials.

Spoilation and clinical performance of monthly vs. three monthly Group II disposable contact lenses.

Twelve subjects were fitted with a high water content, nonionic contact lens (Pilkington Barnes-Hind "Precision UV"), which was either replaced every month for 3 months or worn for 3 consecutive months before replacement occurred. Visual quality, high and low contrast acuity, and comfort were unaltered with either replacement schedule, but overall satisfaction was significantly greater with the shorter replacement schedule (p = 0.04). Front surface wettability revealed a large amount of intersubject variability and was reduced at the 3-month visit with the longer replacement period lenses (p = 0.003). Visible deposits also increased with longer replacement times (p < 0.05). Laboratory-based analytical results showed that both gross lipid and gross extractable protein significantly increased in the 3-month lenses compared with the 1-month lenses, with 44% less lipid accumulation and 60% less protein deposition occurring with the shorter replacement time. The results support the replacement of high water content lenses on a monthly basis.