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Purpose: To examine the spatial profile of macular pigment (MP) and its relationship with serum concentrations of lutein (L) and zeaxanthin (Z) in subjects from a Singapore Chinese population., Methods: In this cross-sectional study, the following details were recorded in 95 healthy subjects: sociodemographic, lifestyle information, body mass index (BMI), visual acuity, MP spatial profile using a macular densitometer, and serum L and Z., Results: The mean (SD) age of the population was 42.40 (± 13) years, ranging from 21 to 68 years. Females demonstrated significantly lower MP optical density (MPOD) than males (MPOD: females = 0.52 ± 0.17; males = 0.61 ± 0.21, P = 0.03). MP spatial profile was typical and atypical with central dip in 68 (85%) and 12 (15%) subjects, respectively. Age and BMI were found to be significant predictors for atypical MP spatial profile (age: odds ratio, OR = 1.06, 95% confidence interval, CI = 1.01-1.13, P = 0.04; BMI: OR = 1.17, 95% CI = 1.01-1.34, P = 0.03). A positive relationship was observed between MPOD and serum concentrations of L and Z, but only the latter relationship reached statistical significance (serum L: r = 0.12, P = 0.30; serum Z: r = 0.26, P = 0.02)., Conclusions: A central dip in MP spatial profile was observed with older age and higher BMI, the two known risk factors for AMD, suggesting that atypical MP spatial profile may be associated with an increased risk of AMD. Further studies with larger sample sizes are required to confirm these observations.

We report a case of suprachoroidal hemorrhage following removal of a releasable suture after combined phacoemulsification-trabeculectomy.

Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing displacement of one DNA strand and formation of a D-loop. Binding of either a T10 PNA or a mixed sequence 15-mer PNA to the transcribed strand of a G-free transcription cassette caused 90 to 100 percent site-specific termination of pol II transcription elongation. When a T10 PNA was bound on the nontranscribed strand, site-specific inhibition never exceeded 50 percent. Binding of PNAs to RNA resulted in site-specific termination of both reverse transcription and in vitro translation, precisely at the position of the PNA.RNA heteroduplex. Nuclear microinjection of cells constitutively expressing SV40 large T antigen (T Ag) with either a 15-mer or 20-mer PNA targeted to the T Ag messenger RNA suppressed T Ag expression. This effect was specific in that there was no reduction in beta-galactosidase expression from a coinjected expression vector and no inhibition of T Ag expression after microinjection of a 10-mer PNA.

Escherichia coli MutH possesses an extremely weak d(GATC) endonuclease that responds to the state of methylation of the sequence (Welsh, K. M., Lu, A.-L., Clark, S., and Modrich, P. (1987) J. Biol. Chem. 262, 15624-15629). MutH endonuclease is activated in a reaction that requires MutS, MutL, ATP, and Mg2+ and depends upon the presence of a mismatch within the DNA. The degree of activation correlates with the efficiency with which a particular mismatch is subject to methyl-directed repair (G-T greater than G-G greater than A-C greater than C-C), and activated MutH responds to the state of DNA adenine methylation. Incision of an unmethylated strand occurs immediately 5' to a d(GATC) sequence, leaving 5' phosphate and 3' hydroxy termini (pN decreases pGpAp-TpC). Unmethylated d(GATC) sites are subject to double strand cleavage by activated MutH, an effect that may account for the killing of dam- mutants by 2-aminopurine. The mechanism of activation apparently requires ATP hydrolysis since adenosine-5'-O-(3-thiotriphosphate) not only fails to support the reaction but also inhibits activation promoted by ATP. The process has no obligate polarity as d(GATC) site incision by the activated nuclease can occur either 3' or 5' to the mismatch on an unmethylated strand. However, activation is sensitive to DNA topology. Circular heteroduplexes are better substrates than linear molecules, and activity of DNAs of the latter class depends on placement of the mismatch and d(GATC) site within the molecule. MutH activation is supported by a 6-kilobase linear heteroduplex in which the mismatch and d(GATC) site are centrally located and separated by 1 kilobase, but a related molecule, in which the two sites are located near opposite ends of the DNA, is essentially inactive as substrate. We conclude that MutH activation represents the initiation stage of methyl-directed repair and suggest that interaction of a mismatch and a d(GATC) site is provoked by MutS binding to a mispair, with subsequent ATP-dependent translocation of one or more Mut proteins along the helix leading to cleavage at a d(GATC) sequence on either side of the mismatch.

To evaluate the substrate specificity of methyl-directed mismatch repair in Escherichia coli extracts, we have constructed a set of DNA heteroduplexes, each of which contains one of the eight possible single base pair mismatches and a single hemimethylated d(GATC) site. Although all eight mismatches were located at the same position within heteroduplex molecules and were embedded within the same sequence environment, they were not corrected with equal efficiencies in vitro. G-T was corrected most efficiently, with A-C, C-T, A-A, T-T, and G-G being repaired at rates 40-80% of that of the G-T mispair. Correction of each of these six mispairs occurred in a methyl-directed manner in a reaction requiring mutH, mutL, and mutS gene products. C-C and A-G mismatches showed different behavior. C-C was an extremely poor substrate for correction while repair of A-G was anomalous. Although A-G was corrected to A-T by the mutHLS-dependent, methyl-directed pathway, repair of A-G to C-G occurred largely by a pathway that is independent of the methylation state of the heteroduplex and which does not require mutH, mutL, or mutS gene products. Similar results were obtained with a second A-G mismatch in a different sequence environment suggesting that a novel pathway may exist for processing A-G mispairs to C-G base pairs. As judged by DNase I footprint analysis, MutS protein is capable of recognizing each of the eight possible base-base mismatches. Use of this method to estimate the apparent affinity of MutS protein for each of the mispairs revealed a rough correlation between MutS affinity and efficiency of correction by the methyl-directed pathway. However, the A-C mismatch was an exception in this respect indicating that interactions other than mismatch recognition may contribute to the efficiency of repair.

A-G mispairs are subject to correction by two distinct pathways in cell-free extracts of Escherichia coli [Su, S.-S., Lahue, R.S., Au, K.G. & Modrich, P. (1988) J. Biol. Chem. 263, 6829-6835; Lu, A.-L. & Chang, D.Y. (1988) Genetics 118, 593-600]. One is the mutHLS-dependent, methyl-directed pathway that recognizes a variety of mismatches and repairs the unmethylated strand of DNA heteroduplexes that are hemimethylated at d(GATC) sequences. The other pathway appears to be specific for A-G mispairs, yields C.G base pairs exclusively, and is independent of the presence of d(GATC) sites. Analyses of cell-free extracts prepared from E. coli mutY strains and isogenic parents have demonstrated that the mutY gene product is involved in the methyl-independent pathway, which converts A-G mispairs to C.G pairs. The specificity of this activity is consistent with the mutator phenotype associated with the mutY locus, which generates G.C----T.A transversions [Nghiem, Y., Cabrera, M., Cupples, C.G. & Miller, J.H. (1988) Proc. Natl. Acad. Sci. USA 85, 2709-2713]. We propose that the mutY product functions at a late stage of a pathway that excludes A-G mispairs during chromosome replication and that involves the function of the mutT gene product. This model suggests that the mutT function acts at an early stage of this pathway to exclude A-G mismatches where the adenine resides on the template DNA strand. A-G mispairs that persist after passage of the replication fork would contain guanine on the template strand and thus be processed to C.G base pairs by the mutY-dependent repair system.

Mercuric ion reductase, a flavoenzyme with an active site redox-active cystine, Cys135-Cys140, is an unusual enzyme that reduces Hg(II) to Hg(0) with stoichiometric NADPH oxidation. To probe the catalytic mechanism, we have constructed two active site Cys to Ser mutations by oligonucleotide-directed mutagenesis. The native and the Cys135, Ser140 and Ser135, Cys140 mutant enzymes are expressed on an overproducing plasmid and purified to homogeneity by a one-step procedure in high yield. The optical spectra of the mutant proteins are distinct, with the Ser135, Cys140 mutant displaying a thiolate-flavin charge-transfer band (Cys140 pKa = 5.2), confirming that Cys140, not Cys135, is in charge-transfer distance both in this mutant and in two electron reduced native enzyme. The native and both mutant proteins are dimers and are precipitated by antibody to native enzyme. Thiol titrations with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicate that both mutants contain three kinetically accessible thiols in both oxidized and reduced states. The native enzyme has two titratable thiols when oxidized and four in the two electron reduced state. The native and two Cys to Ser mutant enzymes show differentiable NADPH-dependent catalytic behavior with Hg(SR)2 (R = CH2CH2OH), Hg(CN)2, DTNB, thio-NADP+, and O2, the most striking of which are the activities toward the Hg(II) complexes and DTNB. Only native enzyme reduces Hg(SR)2. The Ser135, Cys140 enzyme alone shows sustained Hg(CN)2 reduction, whereas the native and Cys135, Ser140 enzymes are rapidly inactivated. DTNB reduction is catalyzed by the native and Cys135, Ser140 enzymes, but not by the Ser135, Cys140 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Mercuric reductase, a flavoenzyme that possess a redox-active cystine, Cys135Cys140, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, we have constructed mutants lacking a redox-active disulfide by eliminating Cys135 (Ala135Cys140), Cys140 (Cys135Ala140), or both (Ala135Ala140). Additionally, we have made double mutants that lack Cys135 (Ala135Cys139Cys140) or Cys140 (Cys135Cys139Ala140) but introduce a new Cys in place of Gly139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. These differences are manifested in a 23-nm range in enzyme-bound FAD lambda max values, an 80-nm range in thiolate to flavin charge-transfer absorbance maxima, and a ca. 100-mV range in FAD reduction potential. Preliminary evidence for the Ala135Cys139Cys140 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala135Cys140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. For these activities, there is a linear correlation between log kappa cat and enzyme-bound FAD reduction potential. In a sensitive Hg(II)-mediated enzyme-bound FADH2 reoxidation assay, all mutant enzymes were able to undergo at least one catalytic event at rates 50-1000-fold slower than that of the wild-type enzyme. We have also observed the reduction of Hg(II) by free FADH2. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. We conclude that the Cys135 and Cys140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.

Mutations in the mutY gene of Escherichia coli confer hypermutability reflecting G.C to T.A transversion mutations and result in a deficiency in methyl-independent G-A to G.C mismatch correction. In the present work, the mutY product has been purified to near homogeneity by virtue of its ability to restore G-A to G.C mismatch correction to cell-free extracts of a mutS mutY strain. The 36-kDa protein renders the strand containing the mispaired adenine labile to base-catalyzed cleavage and sensitive to cleavage by several apurinic/apyrimidinic-site endonucleases, with the sites of strand scission by both agents corresponding to the location of the mismatch. These findings indicate that MutY is a DNA glycosylase that hydrolyzes the glycosyl bond linking the mis-paired adenine to deoxyribose. MutY, a 5'-apurinic/apyrimidinic-site endonuclease, DNA polymerase I, and DNA ligase are sufficient to reconstitute MutY-dependent G-A to G.C repair in vitro.

DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation of widely separated DNA sites. The Escherichia coli methyl-directed reaction has been reconstituted in a purified system consisting of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, along with ATP (adenosine triphosphate), and the four deoxynucleoside triphosphates. This set of proteins can process seven of the eight base-base mismatches in a strand-specific reaction that is directed by the state of methylation of a single d(GATC) sequence located 1 kilobase from the mispair.