Adrienne Boone

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure.

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.

Uptake and release of ciprofloxacin-HCl from conventional and silicone hydrogel contact lens materials.

Objectives. To investigate the uptake and release characteristics of the antibiotic ciprofloxacin-HCl in conventional and silicone hydrogel lenses, and evaluate their potential as therapeutic drug delivery devices. Methods. Nine differing soft contact lens materials were soaked in a 0.3% ciprofloxacin-HCl solution at 34°C. The uptake of the drug into the lenses was measured by the change in concentration over 24 hours using fluorescence spectrophotometry. The lenses were then placed in a buffered saline solution, and the release of the drug from the lenses was also measured using spectrophotometry. Results. The release of drug varied from 0.016 ± 0.004 mg/lens for lotrafilcon A lenses to 0.42 ± 0.03 mg/lens for etafilcon A lenses, with an average of 0.133 mg/lens. The 3 conventional lenses used in the study released a statistically significantly different amount of drug when compared with the silicone hydrogels. The release of drug was very rapid, with drug release reaching a plateau after no more than 10 minutes for the majority of the lenses. The majority of the lenses were able to release enough drug to achieve minimum inhibitory concentration 90 for most resistant ocular pathogens. Ciprofloxacin was found to heavily precipitate on the etafilcon A lenses during the release phase at physiologic pH. Conclusion. While balafilcon A released the most drug from the silicone hydrogel materials, all materials released the drug too quickly to be effective as drug delivery devices.

Uptake and release of dexamethasone phosphate from silicone hydrogel and group I, II, and IV hydrogel contact lenses.

Objectives To investigate the uptake and release kinetics of the synthetic glucocorticoid anti-inflammatory drug dexamethasone into various conventional and silicone hydrogel contact lens materials. Methods Three conventional and six silicone hydrogel lenses were used in this study. A 0.1% dexamethasone solution was formulated and used to dope the various contact lens materials. The uptake and release of the drug was measured using a UV-visible light spectrophotometer at various time points during a period of 24 hr for each phase. Results Statistical analysis showed that all lenses took up a significant amount of dexamethasone. Alphafilcon A and lotrafilcon A showed the greatest uptake of dexamethasone, at 118 ± 10 &mgr;g/lens and 102 ± 11 &mgr;g/lens, respectively, and galyfilcon took up the least amount of drug at 34 ± 6 &mgr;g/lens. The release of the drug from the lens materials was also statistically significant. The majority of the lenses released between 20 and 30 &mgr;g/lens, except for alphafilcon A and lotrafilcon A, which released a statistically different amount of drug when compared with the other lens materials. Alphafilcon A released 65 ± 1.3 &mgr;g/lens, whereas lotrafilcon A slowly released only 11 ± 0.2 &mgr;g/lens. Conclusions Although most of the lenses released enough drug to have anti-inflammatory action, none of the materials released drug for a long enough period of time to be clinically useful as a drug delivery device.

Copper impact on heat shock protein 70 expression and apoptosis in rainbow trout hepatocytes

The mechanism underlying copper hepatotoxicity was investigated in primary cultures of rainbow trout hepatocytes maintained in Leibovitz-15 media. CuSO4 treatment (0, 25, 50, 100 and 200 microM) resulted in a dose-dependent elevation in heat shock protein 70 (hsp70) expression at 24 and 48 h post-exposure. There was no effect of copper (200 microM CuSO4) on hepatotoxicity at 24 h, whereas longer exposures (48 h) resulted in increased lactate dehydrogenase (LDH) leakage and apoptosis, demonstrated by fluorescence nuclear staining and DNA fragmentation. Vitamin C (1 mM), a free radical scavenger, inhibited this copper-induced apoptosis implying a role for reactive oxygen species in copper toxicity. However, no parallel inhibition of either LDH leakage or hsp70 protein expression was observed with vitamin C suggesting that at least two independent mechanisms are involved in the cellular response to copper. Also, copper exposed (24 h) cells were unable to mount an hsp70 response to a standardized heat shock (+15 degrees C for 1 h), even in the presence of vitamin C. Together, these results suggest that hepatotoxicity of copper includes impairment of hsp70 response to subsequent stressors and/or signals, which is crucial for protecting cells from proteotoxicity.

Tear lipocalin and lysozyme in Sjögren and non-Sjögren dry eye.

Purpose. To evaluate the concentration of tear lipocalin, lysozyme, and total protein in Sjogrens Syndrome (SS), non-Sjogrens keratoconjunctivitis sicca (KCS), and non-dry-eyed (NDE) individuals. Methods. Seventy-six subjects were recruited for this study: 25 SS subjects; 25 KCS subjects, and 26 NDE individuals. Symptoms were measured with a visual analogue scale. Tear flow was measured by the Schirmer I test without anesthesia. Tears were collected using an eye wash technique. Total tear protein was quantified using the DC Protein Assay Kit. Tear lipocalin and lysozyme were quantified via Western blotting performed on a Phast System. Results. By definition, the SS and KCS groups both had significantly lower mean Schirmer scores (5.12 ± 5.96 mm and 7.84 ± 7.35 mm) compared with the NDE group (23.83 ± 7.85 mm; p < 0.0001). There was no difference in mean Schirmer scores between SS and KCS groups (p = 0.19). The tear film of the SS group was characterized by significantly reduced (p < 0.0001) total protein and lipocalin concentrations compared with both KCS and NDE groups. No difference between the KCS and NDE groups was found in total protein (p = 0.92) or lipocalin (p = 0.19) concentration. In contrast, the concentration of tear film lysozyme was found to be statistically similar in all three groups examined. No statistically significant correlation was found in any group between mean Schirmer values compared with total protein, lipocalin or lysozyme concentration. Conclusion. Our data demonstrate a biochemical distinction between the Sjogrens group compared with both KCS and control groups, in that both tear lipocalin and total tear protein were significantly reduced. Although correlations were not found between protein measurements and tear flow, a combination of tests including Schirmer I and quantitation of tear film biomarkers may allow for the identification of SS patients without the need for invasive testing.

Ex Vivo Protein Deposition on Bi-Weekly Silicone Hydrogel Contact Lenses

Purpose. This study investigated the protein deposition that occurs on daily wear silicone hydrogel (SH) lenses, after 2 weeks of wear. Methods. A total of 40 subjects were divided into equal groups, based on their habitual SH contact lens [CIBA Vision O2OPTIX (O2); Johnson & Johnson ACUVUE ADVANCE with HYDRACLEAR (ADV); Bausch & Lomb PureVision (PV); CIBA Vision Night & Day (ND)]. A randomized, double-masked, cross-over study was conducted in which subjects wore either their habitual SH material or Johnson & Johnson ACUVUE OASYS with HYDRACLEAR PLUS (OAS) for 2 weeks. At the end of the 2-week period, lenses were collected for analysis of total protein, total lysozyme, and percent denatured lysozyme. Results. Total protein was greatest for PV (33 ± 6 &mgr;g/lens), with other lenses depositing <10 &mgr;g/lens. The PV amount was greater than other materials (p < 0.001), with no significant differences between the other three materials (p > 0.05). Total lysozyme was also greatest for the PV lens (11 ± 3 &mgr;g/lens), with other lenses depositing <2 &mgr;g/lens. The PV amount was greater than all other materials (p < 0.001). ADV and OAS were greater than O2 (p < 0.01) but were not different from each other (p > 0.05). The percentage of lysozyme that was denatured was greatest for ND (90 ± 8%) and lowest for PV (23 ± 10%). The lysozyme extracted from ND and O2 lenses was significantly more denatured than that extracted from the other lens materials (p < 0.001). There were no significant differences for lysozyme activity between ND and O2 (p > 0.05) or between ADV, OAS, and PV (p > 0.05). The amount of denatured lysozyme/lens was <3 &mgr;g/lens for all materials. Lysozyme as a percentage of the total protein deposited ranged from 32 (PV) to 6% (O2). Conclusions. This study confirms that all SH lenses deposit low levels of protein, and that the amount and percentage of denatured lysozyme can vary, depending on the overall surface charge of the material and absence or type of surface treatment.

The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

Hyaluronic acid as an internal wetting agent in model DMAA/TRIS contact lenses

Model silicone hydrogel contact lenses, comprised of N,N-dimethylacrylamide and methacryloxypropyltris (trimethylsiloxy) silane, were fabricated and hyaluronic acid (HA) was incorporated as an internal wetting agent using a dendrimer-based method. HA and dendrimers were loaded into the silicone hydrogels and cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry. The presence and location of HA in the hydrogels was confirmed using X-ray photoelectron spectroscopy and confocal laser scanning microscopy, respectively. The effects of the presence of HA on the silicone hydrogels on hydrophilicity, swelling behavior, transparency, and lysozyme sorption and denaturation were evaluated. The results showed that HA increased the hydrophilicity and the equilibrium water content of the hydrogels without affecting transparency. HA also significantly decreased the amount of lysozyme sorption (p < 0.002). HA had no effect on lysozyme denaturation in hydrogels containing 0% and 1.7% methacrylic acid (MAA) (by weight) but when the amount of MAA was increased to 5%, the level of lysozyme denaturation was significantly lower compared to control materials. These results suggest that HA has great potential to be used as a wetting agent in silicone hydrogel contact lenses to improve wettability and to decrease lysozyme sorption and denaturation.

Chapter 13 Xenobiotic impact on corticosteroid signaling

Publisher Summary This chapter examines the potential sites of action of toxicants on corticosteroid signaling, mostly based on information gathered from literature on mammalian models. In bony fishes, corticosteroids are secreted from the interrenal tissues (analogous to the adrenal cortex) located in the head kidney region. Cortisol stimulation is implicated in a wide array of whole animal changes including osmo- and iono-regulation, respiration, immune responses, reproduction, growth, and metabolism. The chapter presents molecular and biochemical information on the fish corticosteroid receptor (CR), which shows characteristics similar to mammalian CR. The chapter summarizes information on CR signaling and key steps that may be sensitive to direct toxicant impact. The chapter also identifies possible pathways/molecules that may be indirectly involved in CR signaling and sensitive to contaminant impact. Fish corticosteroid receptor summarizes binding studies that have established the receptor characteristics of the glucocorticoid receptor and the mineralocorticoid receptor of CRs in fish.

The effects of hyaluronic acid incorporated as a wetting agent on lysozyme denaturation in model contact lens materials.

Conventional and silicone hydrogels as models for contact lenses were prepared to determine the effect of the presence of hyaluronic acid on lysozyme sorption and denaturation. Hyaluronic acid was loaded into poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate)/TRIS – methacryloxypropyltris (trimethylsiloxy silane) hydrogels, which served as models for conventional and silicone hydrogel contact lens materials. The hyaluronic acid was cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in the presence of dendrimers. Active lysozyme was quantified using a Micrococcus lysodeikticus assay while total lysozyme was determined using 125-I radiolabeled protein. To examine the location of hyaluronic acid in the gels, 6-aminofluorescein labeled hyaluronic acid was incorporated into the gels using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry and the gels were examined using confocal laser scanning microscopy. Hyaluronic acid incorporation significantly reduced lysozyme sorption in poly(2-hydroxyethyl methacrylate) (p < 0.00001) and poly(2-hydroxyethyl methacrylate)/TRIS – methacryloxypropyltris (trimethylsiloxy silane) (p < 0.001) hydrogels, with the modified materials sorbing only 20% and 16% that of the control, respectively. More importantly, hyaluronic acid also decreased lysozyme denaturation in poly(2-hydroxyethyl methacrylate) (p < 0.005) and poly(2-hydroxyethyl methacrylate)/TRIS – methacryloxypropyltris (trimethylsiloxy silane) (p < 0.02) hydrogels. The confocal laser scanning microscopy results showed that the hyaluronic acid distribution was dependent on both the material type and the molecular weight of hyaluronic acid. This study demonstrates that hyaluronic acid incorporated as a wetting agent has the potential to reduce lysozyme sorption and denaturation in contact lens applications. The distribution of hyaluronic acid within hydrogels appears to affect denaturation, with more surface mobile, lower molecular weight hyaluronic acid being more effective in preventing denaturation.