Direct effect of streptozotocin on periodontal ligament cells through myeloid cell leukemia-1.

OBJECTIVE: We hypothesized that streptozotocin (STZ) has a direct impact on periodontal ligament cell (PDL) damage as a potential direct inducer of periodontitis. BACKGROUND: Since diabetes was accepted as one of the risk factors for the development of periodontal disease, various scientific studies have been undertaken in the STZ-induced periodontal disease models. STZ induces ß-cell damage and subsequent diabetes development in vivo. Until now, assessment of the impacts of STZ-induced experimental diabetes on periodontitis has generally been conducted on the fundamental assumption that STZ have no direct action on PDL and its function. However, several recent studies suggest that STZ also directly affect many different biological functions in various tissues or organs. MATERIAL AND METHODS: To assess the apoptotic effects of STZ on PDLs, they were treated with or without STZ at different concentrations. Qualitative estimation of apoptotic cell death was obtained by live/dead assay. The expression levels of apoptosis-related proteins were evaluated by western blot analysis. RESULTS: STZ inhibits growth and induces apoptosis in PDLs in a dose-dependent manner. Furthermore, STZ dramatically induced Mcl-1 downregulation in a proteasome-dependent manner and thereby induced apoptosis of PDLs through the Bak/Bax apoptotic signaling pathway. CONCLUSION: Our results support the hypothesis that suppression of the cellular Mcl-1 levels by STZ may be at least partly attributed to the development of periodontitis in STZ-induced diabetic animal models.

The in vitro apoptotic effects of A248 and A1659, newly synthetic histone deacetylase inhibitors in oral cancer cells.

OBJECTIVES: Histone deacetylase (HDAC) inhibitors represent potential therapeutic agents against various cancers. In this study, we attempt to identify whether newly synthesized HDAC inhibitors, A248 and A1659, can be effective anti-cancer drug candidates for oral cancer. MATERIALS AND METHODS: The anti-cancer activities of A248 and A1659 in MC-3 and HN22 human oral cancer cells were evaluated by 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, Western blot analysis, immunocytochemistry, and small interference RNA (siRNA) technology. RESULTS: A248 and A1659 enhanced histone acetylation and decreased the viability of MC-3 and HN22 cells. A248 and A1659 also induced apoptosis, as evidenced by altered nuclear features and poly(ADP-ribose)polymerase (PARP) cleavage. A248 and A1659 markedly decreased Sp1 expression in a concentration- or time-dependent manner and blocked nuclear translocation of Sp1 protein from the cytosol, which contributed to an increase in p27 expression and a decrease in cyclin D1 expression. Furthermore, the knockdown of Sp1 protein with siRNA caused marked alteration of p27 and cyclin D1 expression to induce apoptosis. The most popular HDAC inhibitor, trichostatin A (TSA) also induced apoptosis and reduced the expression level of Sp1 protein. CONCLUSION: These results suggest that A248 and A1659, two new HDAC inhibitors, may be attractive therapeutic drug candidates for targeting Sp1 in human oral cancer cells.

Apoptotic effect of dibenzylideneacetone on oral cancer cells via modulation of specificity protein 1 and Bax.

OBJECTIVE: Dibenzylideneacetone (DBA), an analogue of curcumin, has been shown to have potential anticancer effects against several cancers. However, the molecular mechanism underlying anticancer activity of DBA has not been well established yet. In this study, we investigated the function and molecular mechanism of DBA in human oral cancer cells. MATERIALS AND METHODS: The growth-inhibitory and apoptotic effects and related signaling pathways of DBA were evaluated using trypan blue exclusion assay, 4'-6-diamidino-2-phenylindole staining, Western blot analysis, siRNA, and reverse transcription-polymerase chain reaction. RESULTS: DBA inhibited cell growth and induced apoptosis, as evidenced by PARP cleavage, activation of caspase-3, and nuclear condensation. DBA also decreased specificity protein 1 (Sp1) expression through facilitating protein degradation. In addition, DBA enhanced the induction of pro-apoptotic protein Bax, resulting in their conformational change, translocation into mitochondrial outer membrane, and its oligomerization. The down-regulation of Sp1 by siRNA targeting Sp1 and mithramycin A increasingly activated Bax to trigger apoptosis. Moreover, DBA-induced growth inhibition and apoptosis in various human oral cancer cell lines were associated with Sp1 down-regulation and induction of Bax. CONCLUSION: These findings suggest that DBA may be a potential anticancer drug candidate to induce apoptosis through down-regulation of Sp1 in human oral cancer.

ß-Phenethyl isothiocyanate induces death receptor 5 to induce apoptosis in human oral cancer cells via p38.

OBJECTIVES: ß-Phenylethyl isothiocyanate (PEITC) has been demonstrated to fight many types of cancers through various molecular pathways. In this study, we focused on its effect on the induction of apoptosis to inhibit cell growth and molecular mechanism in oral cancer. MATERIALS AND METHODS: 3-(4,5-dimethylthiazol-2-yl)-5-(2,4-disulfophenyl)-2-(4 sulfophenyl)-2H-tetrazolium (MTS) assay was used to examine cell viability. The apoptotic effect was investigated using 4'-6-Diamidino-2-phenylindole (DAPI) staining or Western blotting. Inhibitors were used to determine the molecular target and mechanism of PEITC-mediated apoptosis. RESULTS: ß-Phenylethyl isothiocyanate inhibited the growth of HN22 human oral cancer cells and induced caspase-dependent apoptosis in HN22 cells as evidenced by nuclear fragmentation and the activation of caspase 3. It increased cleaved caspase 8, truncated BID, and death receptor 5 (DR5) through the activation of p38 MAPK. This result was confirmed by blockage of PEITC-induced cleavages of Poly(ADP-ribose) Polymerase, caspase-3, caspase-8, and DR5 by p38 MAPK inhibitor, SB203580. We also found that PEITC activated p38 and augmented DR5 to induce apoptosis in other human oral cancer cells. CONCLUSIONS: These results suggest that DR5 is a potential molecular target for PEITC-induced apoptosis in oral cancer via p38 MAPK.

Apoptotic effect of Polygonum Cuspidatum in oral cancer cells through the regulation of specificity protein 1.

OBJECTIVES: The aim of this study was to evaluate the growth inhibitory and apoptosis-inducing effects and mechanisms of Polygonum cuspidatum root in oral cancer cells. MATERIALS AND METHODS: The testing materials were separated by normal-phase silica gel liquid chromatography. The effect of P. cuspidatum root on apoptotsis and its mechanism were performed using 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (MTS) assay, western blot analysis, RT-PCR, promoter assay, and (4'-6-Diamidino-2-phenylindole) (DAPI) staining. RESULTS: The methanol extract of P. cuspidatum (MEPC) inhibited the proliferation of oral cancer cells by inducing caspase-dependent apoptosis. Protein and mRNA expression levels and the transactivation of Specificity protein 1 (Sp1) were markedly decreased in KB cells treated with MEPC. Ethyl acetate fraction (EA) from MEPC was more potent than aqueous fraction (AQ) from MEPC to induce apoptosis. F2, F3, and F4 from EA differentially inhibited the growth of KB cells, and it depends on the amount of Emodin in F2, F3, and F4. Moreover, Emodin inhibited oral cancer cell growth and induced caspase-dependent apoptosis by decreasing Sp1. MEPC also decreased an apoptosis-related downstream target of Sp1 protein, survivin. CONCLUSION: The results from this study strongly suggest that MEPC, its fraction, and Emodin may be potential bioactive materials to cause apoptosis mechanism via the down-regulation of Sp1 in oral cancer cells.

Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft growth.

Developing therapeutics to effectively inhibit the MYC oncoprotein would mark a key advance towards cancer patient care as MYC is deregulated in over 50% of human cancers. MYC deregulation is correlated with aggressive disease and poor patient outcome. Despite strong evidence in mouse models that inhibiting MYC would significantly impact tumour cell growth and patient survival, traditional approaches have not yet yielded the urgently needed therapeutic agents that directly target MYC. MYC functions through its interaction with MAX to regulate gene transcription by binding to E-box DNA response elements of MYC target genes. Here we used a structure-based strategy to design ME47, a small minimalist hybrid protein (MHP) able to disrupt the MAX:E-box interaction/binding and block transcriptional MYC activity. We show that inducing ME47 expression in established tumour xenografts inhibits tumour growth and decreases cellular proliferation. Mechanistically, we show by chromatin immunoprecipitation that ME47 binds to E-box binding sites of MYC target genes. Moreover, ME47 occupancy decreases MYC:DNA interaction at its cognate E-box binding sites. Taken together, ME47 is a prototypic MHP inhibitor that antagonizes tumour cell growth in vitro and in vivo and inhibits the interaction of MYC with DNA E-box elements. These results support ME47's role as a MYC inhibitor and suggest that MHPs provide an alternative therapeutic targeting system that can be used to target transcription factors important in human diseases, including cancer.

Baseline findings in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study.

PURPOSE: To describe the baseline findings in patients enrolled in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study. METHODS: This is a longitudinal observational study of 1209 patients with keratoconus enrolled at 16 clinical centers. Its main outcome measures are corneal scarring, visual acuity, keratometry, and quality of life. RESULTS: The CLEK Study patients had a mean age of 39.29+/-10.90 years with moderate to severe disease, assessed by a keratometric-based criterion (95.4% of patients had steep keratometric readings of at least 45 D) and relatively good visual acuity (77.9% had best corrected visual acuity of at least 20/40 in both eyes). Sixty-five percent of the patients wore rigid gas-permeable contact lens, and most of those (73%) reported that their lenses were comfortable. Only 13.5% of patients reported a family history of keratoconus. None reported serious systemic diseases that had been previously reported to be associated with keratoconus. Many (53%) reported a history of atopy. Fifty-three percent had corneal scarring in one or both eyes. CONCLUSIONS: Baseline findings suggest that keratoconus is not associated with increased risk of connective tissue disease and that most patients in the CLEK Study sample represent mild to moderate keratoconus. Additional follow-up of at least 3 years will provide new information about the progression of keratoconus, identify factors associated with progression, and assess its impact on quality of life.

Capsular opening contraction after continuous curvilinear capsulorhexis and intraocular lens implantation.

PURPOSE: To evaluate the progressive constriction of the anterior capsule opening that can occur after continuous curvilinear capsulorhexis (CCC). SETTING: Kangnam St. Mary's Hospital, Seoul, Korea. METHODS: Changes in the anterior capsule opening after CCC were evaluated in 166 pseudophakic eyes at 1 week and 1 and 3 months postoperatively. The capsular opening diameter was measured with an image analysis system. RESULTS: The capsular opening diameter was reduced by an average of 13.87% 3 months after CCC. There was more dense opacity in the anterior than in the posterior capsule. Lens epithelial cells (LECs) were the main cause of capsule contraction; sex, age, intraocular lens haptic length and haptic material, and CCC size did not have a statistically significant effect on capsule shrinkage (P > .05). Three months after surgery, most eyes with an initial capsular opening diameter of less than 5.5 mm had an opening diameter smaller than 5.0 mm. In most eyes with an initial capsular opening larger than 5.5 mm, the opening remained larger than 5.0 mm. CONCLUSION: Our results suggest that the ideal CCC size is 5.5 to 6.0 mm or larger and that LEC removal is necessary to preserve the pupillary zone and thus prevent progressive capsular opening shrinkage.

Discordance for keratoconus in two pairs of monozygotic twins.

PURPOSE: We present two pairs of monozygotic twins discordant for keratoconus. METHODS: Two pairs of twins, each with one twin with keratoconus, and available family members were examined clinically and with computer-assisted videokeratography. Polymerase chain reaction-based zygosity assays using between nine and 11 unique, anonymous DNA markers were performed on blood obtained from the twins and surviving parents to assess the probability of genetic monozygosity. RESULTS: DNA probes showed a >99% probability that each of the two sets of twins was monozygotic. One twin from each pair had clinically diagnosed keratoconus. The remaining twins were normal by clinical examination and corneal topography. Clinical results for all family members examined were normal except that five of 13 from one family and one of six from the other family demonstrated "suspicious" corneal topography. CONCLUSION: Recent advances in knowledge and understanding of the twinning process suggest that monozygotic twins discordant for keratoconus does not preclude the possibility of a significant genetic component.

HRCT features of acute rejection in patients with bilateral lung transplantation: the usefulness of lesion distribution.

PURPOSE: This study sought to evaluate the high-resolution computed tomography (HRCT) features of acute rejection and to assess the diagnostic accuracy of HRCT for acute rejection considering distribution of lesions in patients with bilateral lung transplantation (BLT). MATERIALS AND METHODS: Between March 2010 and June 2012, 48 transbronchial lung biopsies (TBLBs) and HRCT were performed simultaneously in 26 patients who underwent BLT. We evaluated the presence of ground glass opacity (GGO), consolidation, nodule, bronchial wall thickening, interlobular septal thickening, pleural effusion, atelectasis, bronchiectasis, and cardiomegaly on the HRCT images. The distribution of lesions was analyzed according to bilaterality or upper/lower predominance. Acute rejection was determined on the basis of the pathologic results of TBLB. We evaluated potential correlations of HRCT features with acute rejection, then assessed overall diagnostic accuracy of various HRCT features in combination to diagnose acute rejection in the transplanted lung. RESULTS: Among the 48 TBLBs, 8 were diagnosed as acute rejection (A1, 4 cases; A2, 2 cases; and A3, 2 cases) pathologically. Two A1 rejections and one A2 rejection appeared normal on computed tomography images. Without considering the distribution of lesions, interlobular septal thickening was significantly associated with acute rejection (P = .010) only. Regarding the distribution of lesions on HRCT images, not only interlobular septal thickening but also GGO was significantly associated with acute rejection (P < .05). The sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy of the HRCT scan in the evaluation of acute rejection were 50%, 97.5%, 80%, 90.1%, and 89.6%, when the bilateral GGO and interlobular septal thickening with lower predominance were considered as the positive finding. CONCLUSIONS: HRCT findings considering lesion distribution could be a useful tool in diagnosing acute rejection in patients with BLT.

Activation of estrogen receptor ß reduces blood-brain barrier breakdown following ischemic injury.

Estrogen receptors (ERs) play important roles in estrogen-mediated neuroprotection. However, their effects on blood-brain barrier (BBB) disruption with vasogenic edema after ischemic stroke have not been determined. We evaluated a role for ERß in the brain without effects in the peripheral reproductive organs for the amelioration of vasogenic edema following ischemic stroke. Transient focal ischemic stroke was induced in ovariectomized female C57BL/6 mice (age 10-11weeks) that were treated with the ERß-selective agonist diarylpropionitrile (DPN). BBB breakdown as determined by the extravasation of endogenous immunoglobulin G (IgG), vasogenic edema, and the infarct volume was significantly reduced by DPN compared to vehicle. Protein expressions of endothelial tight junction proteins (occludin and claudin-5) and the water channel protein aquaporin 4 in the ischemic cortex were not changed by DPN. However, protein levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α), a transcription factor that increases VEGF expression, were significantly decreased in the ischemic cortex by DPN. These results suggest that ERß contributes to the reduction of vasogenic edema caused by BBB breakdown via the inhibition of HIF-1α and VEGF following ischemic stroke.

Nanosized titanium dioxide enhanced inflammatory responses in the septic brain of mouse.

Nanosized titanium dioxide (TiO(2)) is used widely in various everyday products and can be applied to the medical field for diagnostic or therapeutic tools. However, its neurobiological responses have not been defined completely in the brain. To evaluate the acute inflammatory response to TiO(2) particles of two different sizes in normal and septic brains, male C57BL/6 mice were given intraperitoneal injections of fine (<1 microm) or ultrafine (21 nm) TiO(2), 30 min after vehicle or lipopolysaccaride (LPS). In the normal brain, neither fine nor ultrafine TiO(2) induced inflammation. However, in the brains of LPS-exposed mice, ultrafine TiO(2) significantly elevated proinflammatory cytokine interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNAs, and IL-1beta protein levels. Also ultrafine TiO(2) increased the levels of reactive oxygen species and activated microglia 24 h after LPS challenge. In BV2 microglial cells stimulated with LPS, ultrafine TiO(2) enhanced TNF-alpha production and augmented nuclear factor-kB binding activity. These findings suggest that nanosized TiO(2) promotes an exaggerated neuroinflammatory responses by enhancing microglial activation in the pre-inflamed brain, in part.

Enzymatic production of trans-free hard fat stock from fractionated rice bran oil, fully hydrogenated soybean oil, and conjugated linoleic Acid.

Rice bran oil (RBO) was fractionated into 2 phases, solid (S-RBO) and liquid (L-RBO), using acetone at -18 degrees C and the weight yield of each S-RBO and L-RBO was 45.5% and 54.5%, respectively. Then, trans-free hard fat was synthesized from trans-free substrate of S-RBO and fully hydrogenated soybean oil (FHSBO) at different molar ratios (S-RBO : FHSBO; 1 : 1, 1 : 1.5, 1 : 2, and 1 : 3) with Lipozyme TL IM lipase (10% of total substrate). Conjugated linoleic acid (CLA, 20% of total substrate) was used as functional fatty acids for the production of trans-free hard fat. After fatty acid analysis, CLA (12.2% to 14.2%) was found on the triacylglycerol (TAG) backbone of the interesterified products along with stearic (37.6% to 49%), palmitic (15% to 17.9%), and oleic acids (13.3% to 19.2%). The interesterified product contained higher level of saturated fatty acid (62.6% to 70.1%) at sn-2 position. Total tocopherols (alpha-, gamma-, and delta-; 1.4 to 2.6 mg/100 g) and phytosterols (campesterol, stigmasterol, and beta-sitosterol; 220.5 to 362.7 mg/100 g) were found in the interesterified products. From DSC results, solid fat contents of the interesterified products (S-RBO : FHSBO 1 : 1, 1 : 1.5, 1 : 2, and 1 : 3) at 25 degrees C were 23.1%, 27%, 30.1%, and 44.9%. The interesterified products consisted mostly of beta' form crystal with a small portion of beta form. The interesterified product (S-RBO : FHSBO 1 : 1.5) was softer than the physical blend but slightly harder than commercial shortenings as measured by texture analyzer. Thus, trans-free hard fat stock, which may have a potential functionality could be produced with various physical properties.

The contribution of the methyl groups on thymine bases to binding specificity and affinity by alanine-rich mutants of the bZIP motif.

We have used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) to short DNA duplexes, in which thymines were replaced with uracils, in order to quantify the contributions of the C5 methyl group on thymines with alanine methyl side chains. We simplified the alpha-helical GCN4 bZIP by alanine substitution: 4A, 11A, and 18A contain four, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. Titration of fluorescein-labeled duplexes with increasing amounts of protein yielded dissociation constants in the low-to-mid nanomolar range for all bZIP mutants in complex with the AP-1 target site (5'-TGACTCA-3'); binding to the nonspecific control duplex was >1000-fold weaker. Small changes of <1 kcal/mol in binding free energies were observed for wild-type bZIP and 4A mutant to uracil-containing AP-1, whereas 11A and 18A bound almost equally well to native AP-1 and uracil-containing AP-1. These modest changes in binding affinities may reflect the multivalent nature of protein-DNA interactions, as our highly mutated proteins still exhibit native-like behavior. These protein mutations may compensate for changes in enthalpic and entropic contributions toward DNA-binding in order to maintain binding free energies similar to that of the native protein-DNA complex.

Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.

Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.

Short, hydrophobic, alanine-based proteins based on the basic region/leucine zipper protein motif: overcoming inclusion body formation and protein aggregation during overexpression, purification, and renaturation.

GCN4 is a yeast transcriptional regulatory protein; its DNA-binding domain is a basic region/leucine zipper (bZIP) structure that comprises a dimer of alpha-helices capable of high-affinity, sequence-specific recognition of the DNA major groove. We are exploiting what nature has evolved by manipulating the bZIP motif as a molecular recognition scaffold; thus we reduced the elegantly minimal bZIP to an even more simplified structure by substitution with alanine residues-hence, a generic, Ala-based, helical scaffold. These Ala-based mutants are unusual proteins for expression as they are short ( approximately 100 amino acids) and hydrophobic (Ala-mutated basic regions, leucine-zipper dimerization domains). Hydrophobicity posed a major problem throughout the expression, isolation, and purification stages; inclusion body formation and protein aggregation were significant hurdles throughout protein production. We describe measures that solved these problems, including use of high concentrations of denaturant in all steps of protein isolation and purification and use of temperature-dependent renaturing techniques to obtain folded, functional protein. Despite these difficulties, we ultimately retrieved 5-10 mg/L of broth of active, correctly folded protein after the complete purification procedure. Homogeneity of the proteins was established by chromatography, electrophoresis, and mass spectrometry. Furthermore, characterization by circular dichroism and DNase footprinting analysis demonstrates that these alanine-based mutants retain the structure and function of the native GCN4 DNA-binding domain. Remarkably, the most heavily mutated protein, containing 24 alanines of 27 total amino acids in the DNA-binding basic region, still binds the AP-1 site, the target of native GCN4.

Short-term repeatability of hand-held keratometry measurements.

PURPOSE: The Collaborative Longitudinal Evaluation of Ethnicity and Refractive Error (CLEERE) Study is a multi-center, observational investigation of ocular component and refractive error development in schoolchildren. Anterior corneal curvature is one of several ocular components which influence refractive status of the eye, and the CLEERE Study uses the Alcon Auto-Keratometer to measure corneal curvature. This report assesses the repeatability of this hand-held instrument. Previous studies have demonstrated the validity of the Alcon Auto-Keratometer; however, none have assessed its repeatability. METHODS: Sixty children were recruited from clinics affiliated with the Southern California College of Optometry and the University of Houston College of Optometry. Two sets of five hand-held keratometry measurements were obtained on the right eye of each child by one investigator at each site using the Alcon Auto-Keratometer. The two sets of measurements were performed at least 10 minutes apart. RESULTS: The mean differences between the two occasions were not significantly different from zero for either the flat or steep corneal curvature measurements regardless of the number of readings taken. The largest improvement in repeatability, primarily for the steep meridian, occurred when the first two readings were averaged. The addition of readings 3, 4, and 5 to the average did not substantially improve repeatability for either meridian. The 95% limits of agreement between the average of two readings on two occasions for the flat and steep meridians were +/-0.28 and +/-0.39 D, respectively. The 95% limits of agreement after two readings were +/-0.28, +/-0.20, and +/-0.24 D for the M, J0, and J45 vectors, respectively. CONCLUSION: The Alcon hand-held keratometer provides a repeatable measure of corneal curvature as demonstrated by short-term repeat agreement within +/-0.50 D. This level of repeatability can be achieved only by manually averaging two consecutive measurements.

Cycloplegia in African-American children.

PURPOSE: The selection of a cycloplegic agent depends on the desired outcome, the characteristics of the patient receiving the drug, and the associated risks. The Orinda Longitudinal Study of Myopia (OLSM) has used 1% tropicamide to assess the ocular components and cycloplegic refractions in a large cohort of predominantly Caucasian children. Although tropicamide has provided adequate cycloplegia and mydriasis for the OLSM cohort, conventional clinical wisdom and scientific investigations have suggested that tropicamide might not produce adequate cycloplegia and mydriasis for subjects with darker iris pigmentation. In this study one drop of 1% tropicamide followed by one drop of 1% cyclopentolate was used to determine their effectiveness in producing adequate cycloplegia and mydriasis for cycloplegic refraction and ocular component measurements in a group of African-American children. METHODS: Nineteen children [age range 5.5 to 15.6 years, mean 8.4 years +/- (SD) 2.5 years] were tested at Family HealthCare of Alabama, Eutaw, AL. Their accommodative responses were measured using a Canon R-1 autorefractor prior to and at 30, 45, and 60 min after instillation of one drop of 0.5% proparacaine, 1% tropicamide (Mydriacyl), and 1% cyclopentolate (Cyclogyl) in both eyes. A target of 20/155 letters in a 4x4 grid positioned behind a +6.50 diopter (D) Badal lens provided accommodative stimuli of 1.00 D, 2.00 D, and 4.00 D. RESULTS: All results are presented as mean +/-1 SD. Pupils, measured from video frames, dilated rapidly and maximally at 30 min after instillation of eye drops (7.3+/-0.5 mm) Predilation, the mean accommodative responses were 0.17+/-0.29 D for the 1.00 D stimulus, 1.01+/-0.40 D for the 2.00 D stimulus, and 2.77+/-0.74 for the 4.00 D stimulus. At 30 min after drop instillation, the responses were 0.07+/-0.14 D for the 1.00 D stimulus, 0.36+/-0.35 D for the 2.00 D stimulus, and 0.77+/-0.61 for the 4.00 D stimulus. Results were very similar at 45 and 60 min after drop instillation. CONCLUSIONS: Combining 1% tropicamide and 1% cyclopentolate was very effective in providing both cycloplegia and mydriasis adequate for ocular biometry and cycloplegic refractions 30 min after drop instillation in African-American children.

Repeatability of refraction and corrected visual acuity in keratoconus. The CLEK Study Group. Collaborative Longitudinal Evaluation of Keratoconus.

PURPOSE: The purpose of the test-retest phase of the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study was to determine the repeatability of the various parts of the CLEK Study protocol. This paper presents the test-retest parameters of the refraction protocol. METHODS: We examined 138 CLEK Study-eligible patients on two occasions (median, 90 days; range, 22 to 268 days). All patients underwent subjective refraction on two occasions, and contact lens over-refractions were performed either over the patient's habitual rigid contact lenses or over a trial rigid contact lens equal in base curve to the steep keratometric reading in nonrigid contact lens wearers. RESULTS: Mean interoccasion differences +/- SD were -0.32 +/- 2.91 D and -0.17 +/- 1.39 D for subjective refraction sphere and cylinder power, respectively, and the mean absolute difference for subjective refraction cylinder axis was 18.1 +/- 20.2 degrees. The mean interoccasion difference +/- SD for high-contrast visual acuity with subjective refraction was 0.38 +/- 10.9 letters correct. Mean interoccasion differences +/- SD were -0.11 +/- 0.81 D and 0.02 +/- 0.67 D for contact lens over-refraction sphere and cylinder power, respectively, and the mean absolute difference for contact lens over-refraction cylinder axis was 11.6 +/- 9.9 degrees. The mean interoccasion difference +/- SD for visual acuity with contact lens over-refraction was 0.50 +/- 5.2 letters correct and 0.71 +/- 6.9 letters correct for high- and low-contrast visual acuity, respectively. CONCLUSIONS: The repeatability of subjective refraction in keratoconus patients is good but somewhat lower than that found in nondiseased eyes. Only 36% of our repeat measures of sphere power from subjective refraction fell within 0.50 D of each other, compared with more than 90% in studies of normal eyes.

Orientation of the putative recognition helix in the DNA-binding domain of Hin recombinase complexed with the hix site.

On the basis of sequence similarity with other known DNA-binding proteins, the DNA-binding domain of Hin recombinase, residues 139-190, is thought to bind DNA by a helix-turn-helix motif. Two models can be considered that differ in the orientation of the recognition helix in the major groove of DNA. One is based on the orientation of the recognition helix found in the 434 repressor (1-69) and lambda repressor-DNA cocrystals, and the other is based on the NMR studies of lac repressor headpiece. Cleavage by EDTA.Fe attached to a lysine side chain (Ser183----Lys183) near the COOH terminus of Hin(139-184) reveals that the putative recognition helix is oriented toward the center of the inverted repeats in a manner similar to that seen in the 434 and lambda repressor-DNA cocrystals.