BACKGROUND

The mechanical stabilization of the cornea in keratoconus may delay progression of this disease. The cross-linking techniques optimized in corneas of enucleated porcine eyes were investigated under in vivo conditions in rabbits to estimate the biocompatibility and duration of the stiffening effect.

METHODS

Twenty-eight rabbits were treated monocularly, the fellow eye serving as control. The epithelium was mechanically removed and 19 eyes were treated with riboflavin plus ultraviolet irradiation (365 nm, 2 mW/cm2) for 45 min and 9 eyes with 0.075% glutaraldehyde for 20 min. After treatment, the eyelids were sutured for 3 days. The healing process was controlled by slit-lamp examination and photographically documented. After 1 month, 20 animals and after 3 months 8 animals were sacrificed, the eyes enucleated, and the stress-strain relation of the corneas measured and compared to the fellow eye.

RESULTS

The epithelium was closed after 4-5 days. The transparency of the corneas remained clear during follow-up, and there were no signs of inflammatory reaction. Stress for a strain of 6% was higher in the treated corneas by a factor of 1.3 +/- 0.66 (P = 0.319) in the glutaraldehyde group and by a factor of 1.6 +/- 0.75 (P = 0.0408) in the riboflavin group at 1 month, and by 1.3 +/- 0.48 (P = 0.07) at 3 months after treatment.

CONCLUSIONS

The cross-linking technique using riboflavin plus UV irradiation is suitable for at least temporarily stiffening the cornea in vivo and seems to be a promising method for conservative treatment of keratectasia.

In developing countries, urban populations are deemed to have better access to a wider variety of foods than their rural counterparts. Yet, data on micronutrient status are scarce and the impact of urban food consumption behaviors on micronutrient adequacy is not well known. The objective of this study was to assess individual micronutrient adequacy of the diet along with food consumption behaviors of women of reproductive age in Ouagadougou, Burkina Faso. A cross-sectional survey of 182 women was conducted in 2 districts of the city. Nutrient intakes were derived from 3 nonconsecutive quantitative 24-h recalls for each woman. Balance in macronutrients was in the range of recommendations except that mean sugar intake was somewhat high. Mean probability of adequacy across 11 micronutrients was low (0.38). The most problematic micronutrients were vitamin B-12 (only 4% of our sample had sufficient intake), folate (12%), riboflavin (13%), and niacin (20%). Higher intakes of organ meat, flesh foods, vitamin A- and vitamin C-rich fruits and vegetables, and legumes and nuts were significantly associated with lower risk of micronutrient inadequacy. Ready-to-eat foods bought outside the home provided 46% of overall energy intake, 52% of fat intake, and 72% of sugar intake but were not associated with micronutrient inadequacy (P>> 0.05). These results highlight the low micronutrient intakes among women of reproductive age in Burkina Faso, even in an urban area.

BACKGROUND

Maize-meal porridge is used for infant feeding in many African countries, including South Africa. A low-cost, finely milled, maize-meal porridge was fortified with beta-carotene, iron, and zinc (100% of recommended dietary allowance), as well as ascorbic acid, copper, selenium, riboflavin, vitamin B-6, vitamin B-12, and vitamin E.

OBJECTIVE

We assessed whether the fortified porridge could reduce anemia and improve the micronutrient status and motor development of infants.

METHODS

Infants aged 6-12 mo (n = 361) were randomly assigned to receive either the fortified or unfortified porridge for 6 mo. Primary outcomes were hemoglobin and serum retinol, zinc, and ferritin concentrations and motor development. Growth was assessed as a secondary outcome. Primary and secondary outcomes were assessed at baseline and 6 mo.

RESULTS

Two hundred ninety-two infants completed the study. The fortified-porridge group had an intervention effect of 9.4 microg/L (95% CI: 3.6, 15.1 microg/L) for serum ferritin and 9 g/L (95% CI: 6, 12 g/L) for hemoglobin concentrations. The proportion of infants with anemia decreased from 45% to 17% in the fortified-porridge group, whereas it remained >40% in the control group. The fortified-porridge group achieved on average 15.5 of the 25 motor development score items, whereas the control group achieved 14.4 items (P = 0.007). Serum retinol concentration showed an inconsistent effect, and no intervention effect was observed for serum zinc concentrations.

CONCLUSIONS

This low-cost fortified porridge can potentially have a significant effect in reducing anemia and improving iron status and motor development of infants in poor settings. The formulation needs some adjustment in terms of zinc fortification.

OBJECTIVE

To determine the repeatability and validity of a self-administered, 175-item food frequency questionnaire (FFQ) in free-living older people and to assess whether these are influenced by cognitive function.

METHODS

189 free-living people aged 64-80 y were recruited from participants in a previous study.

METHODS

To assess repeatability, 102 (52 M, 50 F) participants completed the FFQ on two occasions three months apart. To assess validity, another 87 participants (44 M, 43 F) completed the FFQ and a four-day weighed diet record three months later. 25 nutrients were studied.

RESULTS

For repeatability, Spearman rank correlation coefficients were above 0.35 (p<0.05) for all nutrients. Cohen's weighted Kappa was above 0.4 for all nutrients except starch, riboflavin, retinol, beta-carotene, and calcium. There were no substantial differences in correlation coefficients between sub-groups divided by short-term memory test score. There was no clear pattern for correlation coefficients in sub-groups divided by executive function test score. For validity, the Spearman rank correlation coefficients were above 0.2 (p<0.05) for all nutrients except fat, mono-unsaturated fatty acids, niacin equivalents and vitamin D, and Cohen's weighted kappa was above 0.4 for alcohol and was above 0.2 for 13 other nutrients. Participants in the lowest-score groups of short-term memory and executive function had the lowest median Spearman correlation coefficient.

CONCLUSIONS

The FFQ had reasonable repeatability and validity in ranking nutrient intakes in this population though the results varied between nutrients. Poor short-term memory or executive function may affect FFQ validity in ranking nutrient intakes.

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To demonstrate the antimicrobial properties of riboflavin/UV-A (365 nm) against fungal pathogens.

METHODS

The antimicrobial properties of riboflavin/UV-A (365 nm), with or without previous treatment with amphotericin B, were tested on three groups of fungi selected from severe cases of keratomycosis: Candida albicans, Fusarium sp, and Aspergillus fumigatus. They were tested by using Kirby-Bauer discs with empty disc (control), riboflavin 0.1% alone (R), UV-A alone (UV-A), riboflavin 0.1% and additional UV-A exposure (R+UV-A), amphotericin B alone (A), amphotericin B and riboflavin 0.1% (A+R), amphotericin B and UV-A (A+UV-A), amphotericin B and riboflavin 0.1%, and additional UV-A exposure (A+R+UV-A). The mean growth inhibition zone (GIZ) was measured around the discs.

RESULTS

C. albicans, Fusarium sp, and A. fumigatus did not show any increased GIZ after treatment without previous amphotericin B medication. However, GIZ was significantly greater after pretreatment with amphotericin B and riboflavin/UV-A (A+R+UV-A) for C. albicans (P = 0.0005), Fusarium sp (P = 0.0023) and A. fumigatus (P = 0.0008) compared with A, A+R, and A+UV-A.

CONCLUSIONS

Amphotericin B is believed to interact with fungi membrane sterols to produce aggregates that form transmembrane channels. Given that collagen is one of the principal components of the cornea, it is also probable that amphotericin B may diffuse easily after cross-linking. Previous treatment with amphotericin B allowed riboflavin/UV-A effectiveness against C. albicans, Fusarium sp, and A. fumigatus. This schema might be used in the future for the treatment of keratomycosis.

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(NO2)CH2CH3 + O2 + H2O leads to OHCCH2CH3 + HNO2 + H2O2. In addition to 1-nitropropane, 3-nitro-2-pentanol, 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavin adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavin 5'-phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and N-ethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavin adenine dinucleotide, 1.33 micrometer.

The influence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) on riboflavin and UVA-mediated one-electron oxidation of an aqueous aerated solution of 2'-deoxyguanosine (dGuo) has been studied. Using labeled experiments, we have demonstrated that, despite not being able to detect significant amounts of 8-oxodGuo upon one-electron oxidation of dGuo, 8-oxodGuo is indeed produced but is further rapidly degraded to oxidized nucleosides. Evidence is provided showing that an efficient electron transfer reaction from 8-oxodGuo to the guanine radical cation or rather its deprotonated form occurs, giving rise to the specific decomposition of 8-oxodGuo together with the restitution of dGuo. It could be concluded that 8-oxodGuo efficiently protects dGuo from decomposition by the one-electron oxidation reaction.

In Streptomyces davawensis roseoflavin is synthesized from GTP and ribulose-5-phosphate through riboflavin. As a first step towards the molecular analysis of flavin metabolism in S. davawensis the genes involved in riboflavin biosynthesis were cloned by hybridization of heterologous probes to a genomic library on a high-density colony-array. The genes ribB (riboflavin synthase, alpha-chain; EC 2.5.1.9), ribM (putative membrane protein), ribA (bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase; EC 3.5.4.25) and ribH (lumazine synthase; EC 2.5.1.9) are organized in an operon-like cluster. Northern blot analysis of this cluster revealed two transcripts of 1.7 and 3.1 kb, respectively. The gene ribB was overexpressed in Escherichia coli. The specific riboflavin synthase activity in a cell-free extract of a recombinant strain was 0.246 nmol mg(-1 )min(-1). Overexpression of ribM enhanced the transport of riboflavin in the corresponding recombinant E. coli strain. Furthermore, overexpression of ribM increased roseoflavin sensitivity of E. coli. On another subgenomic fragment a putative S. davawensis ribG gene coding for the missing pyrimidine deaminase/reductase (EC 3.5.4.26 and EC 1.1.1.193) of the riboflavin biosynthetic pathway and ribY coding for a second (monofunctional) GTP cyclohydrolase II were identified.

The vacuolar ATPase subunit A structural gene VMA1 of the biotechnologically important riboflavin overproducer Ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungi. The deduced polypeptide comprises 617 amino acids with a calculated molecular mass of 67.8 kDa. The deduced amino acid sequence is highly similar to that of the catalytic subunits of Saccharomyces cerevisiae (67 kDa), Candida tropicalis (67 kDa), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respectively, and shows about 25% identity to the beta-subunit of the FoF1-ATPase of S. cerevisiae and Schizosaccharomyces pombe. In contrast to S. cerevisiae, however, where disruption of the VMA1 gene was conditionally lethal, and to N. crassa, where viable disruptants could not be isolated, disruption of the VMA1 gene in A. gossypii did not cause a lethal phenotype. Disruption of the AgVMA1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed in the wild type.

The generation of reactive oxygen species (ROS) is a steady-state cellular event in respiring cells. Their production can be grossly amplified in response to a variety of pathophysiological conditions such as inflammation, immunologic disorders, hypoxia, hyperoxia, metabolism of drug or alcohol, exposure to UV or therapeutic radiation, and deficiency in antioxidant vitamins. Uncontrolled production of ROS often leads to damage of cellular macromolecules (DNA, protein, and lipids) and other small antioxidant molecules. A number of major cellular defense mechanisms exist to neutralize and combat the damaging effects of these reactive substances. The enzymic system functions by direct or sequential removal of ROS (superoxide dismutase, catalase, and glutathione peroxidase), thereby terminating their activities. Metal binding proteins, targeted to bind iron and copper ions, ensure that these Fenton metals are cryptic. Nonenzymic defense consists of scavenging molecules that are endogenously produced (GSH, ubiquinols, uric acid) or those derived from the diet (vitamins C and E, lipoic acid, selenium, riboflavin, zinc, and the carotenoids). These antioxidant nutrients occupy distinct cellular compartments and among them, there are active recycling. For example, oxidized vitamin E (tocopheroxy radical) has been shown to be regenerated by ascorbate, GSH, lipoic acid, or ubiquinols. GSH disulfides (GSSG) can be regenerated by GSSG reductase (a riboflavin-dependent protein), and enzymic pathways have been identified for the recycling of ascorbate radical and dehydroascorbate. The electrons that are used to fuel these recycling reactions (NADH and NADPH) are ultimately derived from the oxidation of foods. Sickle cell anemia, thalassemia, and glucose-6-phosphate-dehydrogenase deficiency are all hereditary disorders with higher potential for oxidative damage due to chronic redox imbalance in red cells that often results in clinical manifestation of mild to serve hemolysis in patients with these disorders. The release of hemoglobin during hemolysis and the subsequent therapeutic transfusion in some cases lead to systemic iron overloading that further potentiates the generation of ROS. Antioxidant status in anemia will be examined, and the potential application of antioxidant treatment as an adjunct therapy under these conditions will be discussed.

8 alpha-(O-Tyrosyl)riboflavin has been synthesized by condensation of the copper complex of L-tyrosine with 8 alpha-bromotetraacetylriboflavin. The structure of this synthetic product was proven by absorption and 1H NMR spectroscopy and by chemical degradation, which yielded 1 mol of tyrosine per mol of flavin. The synthetic compound comigrated wtih the (aminoacyl)riboflavin isolated from the p-cresol methylhydroxylase of Pseudomonas putida, and both showed identical absorption and fluorescence spectral properties. 8 alpha-(O-Tyrosyl)riboflavin as well as the flavin-containing decapeptide from p-cresol methylhydroxylase undergoes reductive cleavage to form riboflavin and FAD, respectively, on anaerobic treatment with dithionite. In contrast, the native enzyme, on reduction with dithionite, yields a reduced flavin via a red (anionic) flavosemiquinone intermediate, which remains covalently bound to the protein even under denaturing conditions. 8 alpha-(O-Tyrosyl)riboflavin bound to apoflavodoxin is also not cleaved on reduction with dithionite, but, instead, a blue (neutral) semiquinone of tyrosylriboflavin is generated, which is resistant to further reduction with dithionite. Three p-cresol methylhydroxylases, isolated from different strains of Pseudomonas putida, differing in molecular weight and Km values for substrates, contain the same peptide at the flavin site. These data provide definitive proof for the existence of 8 alpha-(O-tyrosyl)riboflavin in nature.

Recent changes in Texas state regulations of child-care foodservice have resulted in more centers halting meal and snack preparation and requiring parents to provide food from home for their children. In the spring of 2006, sack lunches prepared at home for children attending licensed child-care centers were evaluated based on Dietary Reference Intakes (DRIs) and Child and Adult Care Food Program (CACFP) standards. The study included 3- to 5-year-old children attending full-time child-care centers that required parents to provide lunches. Lunch contents were observed and recorded for 3 consecutive days. A 3-day mean nutrient content was used to determine whether the lunches provided a minimum of 33% of the DRI. The following nutrients were evaluated: energy, carbohydrates, protein, dietary fiber, thiamin, riboflavin, niacin, vitamin C, vitamin A, calcium, iron, zinc, and sodium. Food items were summarized and compared with CACFP standards. More than 50% of the 3-day means provided less than 33% of the DRIs for energy (n=58), carbohydrate (n=59), vitamin A (n=58), calcium (n=49), iron (n=44), and zinc (n=38). Seventy-one of the 74 children (96%) received less than 33% of the DRI for dietary fiber, yet the mean amount of sodium in observed lunches was 114% of the DRI. The observed lunches did not meet the CACFP standards for servings of fruits and vegetables for 157 (71%) or for servings of milk in 178 (80%). Sack lunches sent from home may not regularly provide adequate nutrients for the growth and development of young children. Nutrition education should be provided to parents to ensure that sack lunches sent from home meet children's nutritional needs.

We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer. The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins. In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of the base modifications recognized by Fpg protein. The remaining Fpg-sensitive modifications were not recognized by Ogg1 protein and relatively poor by endonuclease III, but they were relatively good substrates of Ntg1 and Ntg2. In the case of the damage induced by photoexcited riboflavin, the fraction of Fpg-sensitive base modifications identified as 8-oxoGua was only 23%. In contrast to the damage induced by singlet oxygen, the remaining lesions were not only recognized by Ntg1 and Ntg2 proteins and (relatively poor) by endonuclease III, but also by Ogg1 protein. The analysis of the mutations observed after transfection of modified plasmid pSV2gpt into Escherichia coli revealed that all agents induced near exclusively GC->>TA and GC->>CG transversions, the numbers of which were correlated with the numbers of 8-oxoGua residues and Ntg-sensitive modifications, respectively. In conclusion, both singlet oxygen and the type-I photosensitizer riboflavin induce predominantly oxidative guanine modifications other than 8-oxoGua, which most probably give rise to GC->>CG transversions and in which eukaryotic cells are substrates of Ntg1 and Ntg2 proteins.

Ammonium persulfate, a common polymerizing agent for acrylamide gels, can inactivate yeast enolase and produce increased electrophoretic heterogeneity during disc electrophoresis in gels containing 8M urea. The use of riboflavin and light for polymerization or thioglycolate for removal of the persulfate are feasible alternatives.

Iron is an essential element in all living cells. Solubilization, uptake and transport of iron by microorganisms is controlled by highly efficient and specific Fe(3+)-chelating agents named siderophores. However, mechanisms of mobilization of iron from ferrisiderophores are still enigmatic. Here, we demonstrate that Escherichia coli contains a powerful enzymatic system for the reduction of ferrisiderophores. Siderophores have a much lower affinity for ferrous iron, which then can be liberated. This system has been previously purified and characterized as a NAD(P)H:flavin oxidoreductase [Fontecave, M., Eliasson, R. and Reichard, P. (1987) J. Biol. Chem. 262, 12,325-12,331)]. It catalyzes the reduction of free flavins, FMN, FAD or riboflavin by NADH or NADPH. Reduced flavins, in turn, transfer their electrons to physiological ferric complexes: ferrisiderophores, ferric citrate and ferritins. The reaction is inhibited by molecular oxygen and greatly stimulated by Fe(2+)-acceptors such as ferrozine or the iron-free form of ribonucleotide reductase subunit R2. We suggest that the reduction and the mobilization of iron from ferrisiderophores in the cell might be regulated by the presence of physiological ferrous traps such as apoproteins.

BACKGROUND

Methylenetetrahydrofolate reductase (MTHFR; EC 1.7.99.5) supplies the folate needed for the metabolism of homocysteine. A reduction in MTHFR activity, as occurs in the homozygous state for the 677C->>T (so-called thermolabile) enzyme variant (TT genotype), is associated with an increase in plasma total homocysteine (tHcy).

OBJECTIVE

In vitro studies suggest that the reduced activity of thermolabile MTHFR is due to the inappropriate loss of its riboflavin cofactor. We investigated the hypothesis that MTHFR activity in the TT genotype group is particularly sensitive to riboflavin status.

METHODS

We studied tHcy and relevant B-vitamin status by MTHFR genotype in a cross-sectional study of 286 healthy subjects aged 19-63 y (median: 27 y). The effect of riboflavin status was examined by dividing the sample into tertiles of erythrocyte glutathionine reductase activation coefficient, a functional index of riboflavin status.

RESULTS

Lower red blood cell folate (P = 0.0001) and higher tHcy (P = 0.0082) concentrations were found in the TT group than in the heterozygous (CT) or wild-type (CC) groups. However, these expected relations in the total sample were driven by the TT group with the lowest riboflavin status, whose mean tHcy concentration (18.09 micromol/L) was almost twice that of the CC or CT group. By contrast, adequate riboflavin status rendered the TT group neutral with respect to tHcy metabolism.

CONCLUSIONS

The high tHcy concentration typically associated with homozygosity for the 677C->>T variant of MTHFR occurs only with poor riboflavin status. This may have important implications for governments considering new fortification policies aimed at the prevention of diseases for which this genotype is associated with increased risk.

Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B(2)). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein's fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host.

OBJECTIVE

Syphilis remains a public health problem, but very little is known about the causative bacterium. This is because Treponema pallidum still cannot be cultured in the laboratory. Rather, T. pallidum must be cultivated in laboratory rabbits, a restriction that poses many insurmountable experimental obstacles. Approaches to learn more about the structure and function of T. pallidum's cell envelope, which is both the physical and functional interface between T. pallidum and its human host, are severely limited. One approach for elucidating T. pallidum's cell envelope has been to determine the three-dimensional structures of its membrane lipoproteins, molecules that serve many critical survival functions. Herein, we describe a previously unknown transport system that T. pallidum uses to import riboflavin, an essential nutrient for the organism's survival. Moreover, we found that this transport system is present in other pathogenic spirochetes. This is the first description of this new type of bacterial riboflavin transport system.

OBJECTIVE

To evaluate riboflavin/ultraviolet-A (UVA) as an adjunct treatment for infectious keratitis.

METHODS

This prospective, dual-center, interventional case series included cases of infectious keratitis that were treated by instilling riboflavin 0.1% solution for 30 minutes to saturate the cornea, followed by exposure to 365-nm UVA light (3 mW/cm(2)) for 15 to 45 minutes, with continued instillation of riboflavin. Eyes continued on standard antibiotic treatment. The primary outcome measures were the times to resolution of the infiltrate and the epithelial defect.

RESULTS

Forty patients aged 14 to 86 years were enrolled. Seven (18%) eyes had a previous keratoplasty. Bacterial species were identified in 24 eyes, fungal in 7, protozoan in 2, viral in 1, and no organism in 6. The maximum infiltrate diameter ranged from 1 to 12 mm and the epithelial defect diameter was 0 to 8 mm before treatment. In 6 cases (2 bacterial, 3 fungal, and 1 without growth), the keratitis did not resolve successfully and the eye received a penetrating keratoplasty (PK). In 1 eye with prior PK, the infection resolved following treatment, but a regraft was required to address perforation of the PK incision.

CONCLUSIONS

Riboflavin/UVA should be avoided in eyes with prior herpes simplex but otherwise posed no obvious safety risk in this series and appeared to be most effective when the infection depth was limited. The success rate was higher for bacterial infections than fungal infections. Randomized studies against antibiotics alone are needed to further evaluate efficacy.

OBJECTIVE

Riboflavin/ultraviolet A (UVA) cross-linking (CXL) of corneal collagen is a novel method of stabilizing corneal mechanical properties and preventing progression of keratectasias. This study was conducted to investigate whether CXL influences ablation rate, flap thickness, and refractive results of excimer laser procedures ex vivo.

METHODS

Corneal epithelium was removed from enucleated porcine eyes, and CXL was performed with riboflavin 0.1% and UVA radiation (365 nm, 3 mW/cm(2)) for 30 minutes. Control eyes received epithelial abrasion only. Diffusion of riboflavin through the cornea was assessed by using infrared-excited, two-photon microscopy of riboflavin autofluorescence, combined with second-harmonic generation of fibrillar collagen. During phototherapeutic keratectomy, corneal thickness was measured by optical coherence pachymetry. During LASIK for myopia, the flap thickness of microkeratome cuts was measured and the induced refractive change assessed by Placido topography. Data were analyzed by Shapiro-Wilk test and Student's t-test.

RESULTS

Multiphoton imaging showed a rapid (30-minute) and even distribution of riboflavin throughout the corneal stroma. No difference in ablation rate was measured in treated and untreated corneas (P = 0.90). Mean flap thickness was increased by 44% in cross-linked corneas (P < 0.01). After LASIK for myopia of 4 to 25 D, the mean corneal refractive change was reduced in CXL-treated eyes by 20.1% (P < 0.05). This effect was less pronounced in thinner flaps.

CONCLUSIONS

CXL reduces the amount of refractive change after LASIK for myopia. Although the laser ablation rate is unaffected, CXL results in an increased flap thickness. This study suggests the need for adjustment of microkeratome and laser parameters for LASIK after CXL and indirectly endorses the theory of a direct stiffening effect of CXL.

The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na(+) across the bacterial membrane. The Na(+)-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na(+)-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na(+)-NQR is discussed.

Near-ultraviolet radiation was found to be lethal for mammalian cells in Dulbecco's modified Eagle's medium without serum or phenol red. Irradiation of the cells with near-ultraviolet light while the cells were in phosphate-buffered-saline abolished the lethal effect. When only the medium was irradiated followed by the addition of unirradiated cells and serum, the cells were still killed. The photoactive components of the medium for this effect were riboflavin, tryptophan, and tyrosine. When riboflavin was deleted from the medium being irradiated and added later, almost no killing was detected. Irradiation of salt solution of riboflavin and tryptophan or riboflavin and tyrosine, resulted in cell killing. Little or no killing resulted when riboflavin, tryptophan, or tyrosine was irradiated singly. The formation of photoproducts toxic for mammalian cells appears to involve photodynamic action. Experiments utilizing Dulbecco's or similar media without proper controls may produce anomalous results from light illuminating the laboratory.