We have newly identified rat riboflavin transporter 2 (rRFT2) and its human orthologue (hRFT2), and carried out detailed functional characterization of rRFT2. The mRNA of rRFT2 was highly expressed in jejunum and ileum. When transiently expressed in human embryonic kidney (HEK) 293 cells, rRFT2 could transport riboflavin efficiently. Riboflavin transport mediated by rRFT2 was Na(+)-independent but moderately pH-sensitive, being more efficient in acidic conditions than in neutral and basic conditions. Kinetic analysis indicated that rRFT2-mediated riboflavin transport was saturable with a Michaelis constant (K(m)) of 0.21 microM. Furthermore, it was specifically and strongly inhibited by lumiflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and to a lesser extent by amiloride. Such ability to transport riboflavin in a specific manner, together with its high expression in the small intestine, indicates that RFT2 may play a role in the intestinal absorption of riboflavin.

Pregnant women in developing countries are vulnerable to multiple micronutrient deficiencies. We investigated their prevalence and seasonal variation as part of a baseline assessment in a population-based, maternal micronutrient supplementation trial conducted in the rural Southeastern plains of Nepal. Serum concentrations of 11 micronutrients were assessed in 1165 pregnant women in the 1st trimester before supplementation. Using defined cutoff values, the prevalence of deficiencies of vitamins A, E, and D were 7, 25, and 14%, respectively. Nearly 33% of the women were deficient in riboflavin, and 40 and 28% had serum vitamin B-6 and B-12 deficiencies, respectively. Only 12% of the women were folate deficient, but 61% were zinc deficient. The prevalence of low serum iron concentration was 40%, and 33% were anemic (hemoglobin < 110 g/L). Multiple micronutrient deficiencies were common among pregnant women. Over 10% of the pregnant women were both anemic and deficient in B-complex vitamins, whereas 22% of women were both anemic and zinc deficient. Only 4% of women had no deficiency, whereas approximately 20% of the women had 2, 3, or 4 deficiencies. Almost 18% of women had>>/=5 deficiencies. Micronutrient status varied by season; it was generally best during the winter months, except for serum vitamin D concentration, which peaked during the hot summer and monsoon months. Women in rural South Asia are likely to begin a pregnancy with multiple micronutrient deficiencies that may vary with seasonality in micronutrient-rich food availability.

Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment in vitro significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.

BACKGROUND

We were able to show a significant increase in corneal stiffness of rabbit and porcine eyes after combined riboflavin/UVA-induced collagen cross-linking. In this study,we tried to treat keratoconus patients with this method to stop the progression of corneal ectasia.

METHODS

We treated 16 eyes of 15 patients with progressive keratoconus and mostly moderate keratectasia (48-56 dpt). After removal of the epithelium (7 mm X), riboflavin solution was applied on the cornea, which was irradiated with UVA (370 nm,3 mW/cm(2)) at a distance of 1 cm for 30 min.Post-operative follow-up controls were conducted every 3 months in the first year and then every 6 months, always including visual acuity testing, corneal topography and measurements of endothelial cell density. The follow-up time was between 1 and 3 years.

RESULTS

Progression of keratectasia was stopped in all patients. Best corrected visual acuity and the maximal keratometry values improved slightly in about 50% of the cases. In all patients corneal transparency, the degree of keratectasia registered by corneal topography and the density of endothelial cells remained unchanged within the follow-up time. No negative side-effects were observed.

CONCLUSIONS

Our results show that collagen cross linking might be a useful conservative treatment modality to stop the progression of keratoconus. By this means the need for keratoplasty might be significantly reduced. Given the simplicity of the technique and minimal costs of the treatment it might also be well suited for developing countries.Further studies are envisaged to exclude long-term side effects and to evaluate the long term durability of the mechanical stiffness effect.

OBJECTIVE

To model the photochemical kinetics of corneal cross-linking with riboflavin (Rf) and confirm the model through measured oxygen concentration experiments under varying energy input conditions by UV-A irradiance and temperature modulation in ex vivo porcine cornea.

METHODS

A theoretical model was developed to describe the corneal cross-linking photochemical kinetics of Rf. After instillation with drops of Rf solution in distilled water, de-epithelialized porcine corneas were exposed to 365-nm ultraviolet light (UV-A) under varying irradiance and temperature. Oxygen concentration in the cornea at a known depth was monitored during UV-A illumination with a dissolved oxygen fiberoptic microsensor. Data from the oxygen experiments were used to confirm the model.

RESULTS

On the basis of the known chemical reactions and diffusion rates of Rf and oxygen into the cornea, the authors developed a theoretical model consistent with corneal oxygen consumption experimental results during UV-A irradiation under different conditions. Oxygen concentration in the cornea is modulated by UV-A irradiance and temperature and quickly decreased at the beginning of UV-A exposure. The time-dependence of both Type-I and Type-II photochemical mechanisms in corneal cross-linking with Rf are discussed.

CONCLUSIONS

Using a chemical kinetics modeling approach, the authors developed a simple model that is in agreement with their experimental results on oxygen consumption in the cornea during corneal cross-linking with Rf. It is suggested that the main photochemical kinetics mechanism is the direct interaction between Rf triplets and reactive groups of corneal proteins, which leads to the cross-linking of the proteins mainly through radical reactions.

A 21-year-old woman had crosslinking for keratoconus in the right eye; the left eye was scheduled for penetrating keratoplasty. Five days postoperatively, she presented with geographic epithelial keratitis and iritis. Analysis of tear samples by polymerase chain reaction confirmed the diagnosis. The patient was treated with oral steroids and acyclovir, with significant improvement. Two months postoperatively, the visual acuity was improved and there was no evidence of herpetic disease recurrence. Crosslinking can induce herpetic keratitis with iritis even in patients with no history of herpetic disease. Early diagnosis and proper treatment are essential for a favorable outcome.

Folates are carriers of one-carbon units and are metabolized by 5,10-methylenetetrahydrofolate reductase (MTHFR) and other enzymes that use <em>riboflavin</em>, cobalamin, or vitamin B6 as cofactors. These B vitamins are essential for the remethylation and transsulfuration of homocysteine, which is an important intermediate in one-carbon metabolism. We studied the MTHFR 677C->>T polymorphism and B vitamins as modulators of one-carbon metabolism in 10,601 adults from the Norwegian Colorectal Cancer Prevention (NORCCAP) cohort, using plasma total homocysteine (tHcy) as the main outcome measure. Mean concentrations of plasma tHcy were 10.4 micromol/liter, 10.9 micromol/liter, and 13.3 micromol/liter in subjects with the CC (51%), CT (41%), and TT (8%) genotypes, respectively. The MTHFR 677C->>T polymorphism, folate, <em>riboflavin</em>, cobalamin, and vitamin B6 were independent predictors of tHcy in multivariate models (P<.001), and genotype effects were strongest when B vitamins were low (P<or=.006). Conversely, the MTHFR polymorphism influenced B vitamin effects, which were strongest in the TT group, in which the estimated tHcy difference between subjects with vitamin concentrations in the lowest compared with the highest quartile was 5.4 micromol/liter for folate, 4.1 micromol/liter for <em>riboflavin</em>, 3.2 micromol/liter for cobalamin, and 2.1 micromol/liter for vitamin B6. Furthermore, interactions between B vitamins were observed, and B vitamins were more strongly related to plasma tHcy when concentrations of other B vitamins were low. The study provides comprehensive data on the MTHFR-B vitamin network, which has major effects on the transfer of one-carbon units. Individuals with the TT genotype were particularly sensitive to the status of several B vitamins and might be candidates for personalized nutritional recommendations.

The status of water- and fat-soluble vitamins was prospectively evaluated in 23 patients (13 men, 10 women, mean age 33 +/- 3 yr) admitted to the hospital with acute or subacute attacks of inflammatory bowel disease. Protein-energy status was also assessed by means of simultaneous measurement of triceps skinfold thickness, mid-arm muscle circumference, and serum albumin. Fifteen patients (group A) had extensive acute colitis (ulcerative or Crohn's colitis), and eight cases (group B) had small bowel or ileocecal Crohn's disease. Eighty-nine healthy subjects (36 men, 53 women, mean age 34 +/- 2 yr) acted as controls. In both groups of patients, the levels of biotin, folate, beta-carotene, and vitamins A, C, and B1 were significantly lower than in controls (p less than 0.01). Plasma levels of vitamin B12 were decreased only in group B (p less than 0.01), whereas riboflavin was lower in group A (p less than 0.01). The percentage of patients at risk of developing hypovitaminosis was 40% or higher for vitamin A, beta-carotene, folate, biotin, vitamin C, and thiamin in both groups of patients. Although some subjects had extremely low vitamin values, in no case were clinical symptoms of vitamin deficiency observed. Only a weak correlation was found between protein-energy nutritional parameters and vitamin values, probably due to the small size of the sample studied. The pathophysiological and clinical implications of the suboptimal vitamin status observed in acute inflammatory bowel disease are unknown. Further studies on long-term vitamin status and clinical outcome in these patients are necessary.

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B(2)) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655-5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (K(d) for riboflavin is 0.6 nM), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure.

OBJECTIVE

Understanding corneal biomechanics is important to refractive or therapeutic corneal treatments. The authors studied the corneal response to variable intraocular pressure (IOP) in porcines eyes after UV collagen cross-linking (CXL), in comparison with untreated eyes.

METHODS

Twenty-three enucleated eyes were treated with standard CXL conditions (365 nm, 3 mW, 30 minutes), and 15 contralateral eyes served as control. Eyes (within a humidity- and temperature-monitored wet chamber) were measured by Scheimpflug corneal three-dimensional topographer. Images were obtained automatically while IOP either remained constant (14 eyes) or increased (24 eyes) by 40 mm Hg and then decreased (4-mm Hg steps). Measurements were performed immediately after treatment and 24 hours later. Corneal geometry was analyzed as a function of IOP, and whole globe stress-strain curves were calculated.

RESULTS

Instillation of riboflavin-dextran solution reduced corneal thickness (by 281 +/- 5 microm). Cross-linking produced a 1.54x reduction in corneal thinning and 2.8x reduction in corneal apical rise with increased IOP. Anterior and posterior cornea flattened with increased IOP (less flattening in CXL eyes) and became steeper with decreased IOP. The horizontal meridian flattened significantly (P < 0.01) more than the vertical meridian. Young's modulus was higher in cross-linked eyes (1.096 +/- 0.30 kN/m(2)) than in non-cross-linked eyes (0.692 +/- 0.30 kN/m(2)). Hysteresis in nontreated eyes was also larger than in cross-linked eyes.

CONCLUSIONS

Cross-linking stiffened porcine corneas significantly. Both experimental data and stress-strain analysis are valuable for finite element models to improve understanding of CXL and its predictability. Although differences are expected between human corneas in vivo and porcine corneas ex vivo, the results are consistent with clinical data found in patients. The apparent biomechanical anisotropy of pig corneas must be confirmed in humans.

The noninvasive estimation of in vivo mechanical properties of cornea is envisioned to find several applications in ophthalmology. Such high-resolution measurements of local cornea stiffness could lead to a better anticipation and understanding of corneal pathologies such as Keratoconus. It could also provide a quantitative evaluation of corneal biomechanical response after corneal refractive surgeries and a tool for evaluating the efficacy of new cornea treatments such as cornea transplant using femtosecond laser or therapy based on Riboflavin/UltraViolet-A Corneal Cross Linking (UVA CXL). In the very important issue of glaucoma diagnosis and management, the fine tuning corneal elasticity measurement could also succeed to strongly correlate the applanation tonometry with the "true" intra-ocular pressure (IOP). This initial investigation evaluates the ability of ultrafast and high-resolution ultrasonic systems to provide a real-time and quantitative mapping of corneal viscoelasticity. Quantitative elasticity maps were acquired ex vivo on porcine cornea using the supersonic shear imaging (SSI) technique. A conventional 15 MHz linear probe was used to perform conventional ultrasonic imaging of the cornea. A dedicated ultrasonic sequence combines the generation of a remote palpation in the cornea and ultrafast (20,000 frames/s) ultrasonic imaging of the resulting corneal displacements that evolve into a shear wave propagation whose local speed was directly linked to local elasticity. A quantitative high-resolution map (150 microm resolution) of local corneal elasticity can be provided by this dedicated sequence of ultrasonic insonifications. Quantitative maps of corneal elasticity were obtained on ex vivo freshly enucleated porcine corneas. In the cornea, a quite homogenous stiffness map was found with a 190 kPa +/- 32 kPa mean elasticity. The influence of photodynamic Riboflavin/UVA induced CXL was measured. A significant Young's modulus increase was obtained with a mean 890 kPa +/- 250 kPa posttreatment Young's modulus (460% increase), located in the anterior part of the cornea. Simulations based on 3-D time domain finite differences simulation were also performed and found to be in good agreement with ex vivo experiments. The SSI technique can perform real-time, noninvasive, high-resolution, and quantitative maps of the whole corneal elasticity. This technique could be real time and straightforward adapted for a very wide field of in vivo investigations.

BACKGROUND

Increased westernization with Japanese migration to the U. S. in the early 20(th) century is thought to have altered the risk of cardiovascular disease. Whether similar effects include changes in the risk of Parkinson's disease (PD) is not clear. This report describes the relations between environmental, life-style, and physical attributes and the incidence of PD that have been observed in the Honolulu-Asia Aging Study.

METHODS

Beginning in 1965, environmental, life-style, and physical attributes were recorded at selected examinations in a cohort of 8,006 Japanese-American men. Subjects were followed for clinical PD.

RESULTS

During 30 years of follow- up, PD was observed in 137 men. Overall incidence (7.1/10,000 person-years) was generally higher than in Asia and similar to rates observed in Europe and the U. S. Precursors of PD included constipation, adiposity, years worked on a sugar or pineapple plantation, years of exposure to pesticides, and exposure to sugar cane processing. Factors showing an inverse association with PD included coffee intake and cigarette smoking. Among dietary factors, carbohydrates increased the risk of PD while the intake of polyunsaturated fats appeared protective. Total caloric intake, saturated and monounsaturated fats, protein, niacin, riboflavin, beta-carotene, vitamins A, B, and C, dietary cholesterol, cobalamin, alpha-tocopherol, and pantothenic acid showed no clear relation with clinical PD.

CONCLUSIONS

Findings suggest that several environmental, life-style, and physical attributes appear to be precursors of PD. Whether patterns of precursors can be used to identify individuals at high risk of future PD or can broaden the scope of early interventions or recruitment into neuroprotective trials warrants further study.

OBJECTIVE

To assess the clinical results of transepithelial collagen crosslinking (CXL) in patients 26 years and younger with progressive keratoconus suitable for epithelium-off (epi-off) CXL.

METHODS

Department of Ophthalmology, Siena University Hospital, Siena, Italy.

METHODS

Prospective case series.

METHODS

The study included 26 eyes (26 patients) treated by transepithelial (epithelium-on) CXL. The mean age was 22 years (range 11 to 26 years) (10 younger than 18 years; 16 between 19 years and 26 years). Preoperative and postoperative examinations included uncorrected (UDVA) and corrected (CDVA) distance visual acuities, simulated maximum keratometry (K), coma and spherical aberration, and corneal optical coherence tomography optical pachymetry. The solution for transepithelial CXL (Ricrolin TE) comprised riboflavin 0.1%, dextran 15.0%, trometamol (Tris), and ethylenediaminetetraacetic acid. Ultraviolet-A treatment was performed with the Caporossi Baiocchi Mazzotta X Linker Vega at 3 mW/cm(2).

RESULTS

After relative improvement in the first 3 to 6 months, the UDVA and CDVA gradually returned to baseline preoperative values. After 12 months of stability, the simulated maximum K value worsened at 24 months. Coma aberration showed no statistically significant change. Spherical aberration increased at 24 months. Pachymetry showed a progressive, statistically significant decrease at 24 months. Fifty percent of pediatric patients were retreated with epi-off CXL due to significant deterioration of all parameters after 12 months of follow-up.

CONCLUSIONS

Functional results after transepithelial CXL showed keratoconus instability, in particular in pediatric patients 18 years old and younger; there was also functional regression in patients between 19 years and 26 years old after 24 months of follow-up. mentioned.

OBJECTIVE

The aim of the National Food Consumption Survey (NFCS) in South Africa was to determine the nutrient intakes and anthropometric status of children (1-9 years old), as well as factors that influence their dietary intake.

METHODS

This was a cross-sectional survey of a nationally representative sample of all children aged 1-9 years in South Africa. A nationally representative sample with provincial representation was selected using 1996 Census information.

METHODS

Of the 3120 children who were originally sampled data were obtained from 2894, a response rate of 93%.

METHODS

The sociodemographic status of each household was assessed by a questionnaire. Dietary intake was assessed by means of a 24-hour recall and a food-frequency questionnaire from the caregivers of the children. Food purchasing practices were determined by means of a food procurement questionnaire. Hunger was assessed by a modified hunger scale questionnaire. Nutritional status was determined by means of anthropometric measurements: height, weight, head circumference and arm circumference.

RESULTS

At the national level, stunting (height-for-age below minus two standard deviations (< -2SD) from the reference median) was by far the most common nutritional disorder, affecting nearly one in five children. The children least affected (17%) were those living in urban areas. Even with regard to the latter, however, children living in informal urban areas were more severely affected (20%) compared with those living in formal urban areas (16%). A similar pattern emerged for the prevalence of underweight (weight-for-age < -2SD), with one in 10 children being affected at the national level. Furthermore, one in 10 (13%) and one in four (26%) children aged 1-3 years had an energy intake less than half and less than two-thirds of their daily energy needs, respectively. For South African children as a whole, the intakes of energy, calcium, iron, zinc, selenium, vitamins A, D, C and E, riboflavin, niacin, vitamin B6 and folic acid were below two-thirds of the Recommended Dietary Allowances. At the national level, data from the 24-hour recalls indicated that the most commonly consumed food items were maize, sugar, tea, whole milk and brown bread. For South African children overall, one in two households (52%) experienced hunger, one in four (23%) were at risk of hunger and only one in four households (25%) appeared food-secure.

CONCLUSIONS

The NFCS indicated that a large majority of households were food-insecure and that energy deficit and micronutrient deficiencies were common, resulting in a high prevalence of stunting. These results were used as motivation for the introduction of mandatory fortification in South Africa.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

In recent years, a population of unconventional T cells called 'mucosal-associated invariant T cells' (MAIT cells) has captured the attention of immunologists and clinicians due to their abundance in humans, their involvement in a broad range of infectious and non-infectious diseases and their unusual specificity for microbial riboflavin-derivative antigens presented by the major histocompatibility complex (MHC) class I-like protein MR1. MAIT cells use a limited T cell antigen receptor (TCR) repertoire with public antigen specificities that are conserved across species. They can be activated by TCR-dependent and TCR-independent mechanisms and exhibit rapid, innate-like effector responses. Here we review evidence showing that MAIT cells are a key component of the immune system and discuss their basic biology, development, role in disease and immunotherapeutic potential.

This study describes food consumption patterns in Canada according to the types of food processing using the Nova classification and investigates the association between consumption of ultra-processed foods and the nutrient profile of the diet. Dietary intakes of 33,694 individuals from the 2004 Canadian Community Health Survey aged 2 years and above were analyzed. Food and drinks were classified using Nova into unprocessed or minimally processed foods, processed culinary ingredients, processed foods and ultra-processed foods. Average consumption (total daily energy intake) and relative consumption (% of total energy intake) provided by each of the food groups were calculated. Consumption of ultra-processed foods according to sex, age, education, residential location and relative family revenue was assessed. Mean nutrient content of ultra-processed foods and non-ultra-processed foods were compared, and the average nutrient content of the overall diet across quintiles of dietary share of ultra-processed foods was measured. In 2004, 48% of calories consumed by Canadians came from ultra-processed foods. Consumption of such foods was high amongst all socioeconomic groups, and particularly in children and adolescents. As a group, ultra-processed foods were grossly nutritionally inferior to non-ultra-processed foods. After adjusting for covariates, a significant and positive relationship was found between the dietary share of ultra-processed foods and the content in carbohydrates, free sugars, total and saturated fats and energy density, while an inverse relationship was observed with the dietary content in protein, fiber, vitamins A, C, D, B6 and B12, niacin, thiamine, riboflavin, as well as zinc, iron, magnesium, calcium, phosphorus and potassium. Lowering the dietary share of ultra-processed foods and raising consumption of hand-made meals from unprocessed or minimally processed foods would substantially improve the diet quality of Canadian.

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., Jörnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.

Curcumin, a naturally occurring phytochemical responsible for the colour of turmeric shows a wide range of pharmacological properties including antioxidant, anti-inflammatory and anti-cancer effects. We have earlier shown that curcumin in the presence of Cu(II) causes strand cleavage in DNA through generation of reactive oxygen species, particularly the hydroxyl radical. Thus, curcumin shows both antioxidant as well as pro-oxidant effects. In order to understand the chemical basis of various biological properties of curcumin, we have studied the structure-activity relationship between curcumin and its two naturally occurring derivatives namely demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC). Curcumin was found to be the most effective in the DNA cleavage reaction and a reducer of Cu(II) followed by dmC and bdmC. The rate of formation of hydroxyl radicals by the three curcuminoids also showed a similar pattern. The relative antioxidant activity was examined by studying the effect of these curcuminoids on cleavage of plasmid DNA by Fe(II)-EDTA system (hydroxyl radicals) and the generation of singlet oxygen by riboflavin. The results indicate that curcumin is considerably more active both as an antioxidant as well as an oxidative DNA cleaving agent. The DNA cleavage activity is the consequence of binding of Cu(II) to various sites on the curcumin molecule. Based on the present results, we propose three binding sites for Cu(II). Two of the sites are provided by the phenolic and methoxy groups on the two benzene rings and the third site is due to the presence of 1,3-diketone system between the rings. Furthermore, both the antioxidant as well as pro-oxidant effects of curcuminoids are determined by the same structural moieties.

Differential cDNA display and quantitative RT-PCR suggested that the riboflavin synthase complex of the aphid endosymbiont, Buchnera, is active only when the symbiotic system is maintained and well organized in young hosts. Since this finding suggested the provision of riboflavin by Buchnera, we examined the effect of dietary riboflavin on the performance of symbiotic and aposymbiotic aphids using chemically-defined diets. Our results indicate: (1) dietary riboflavin is slightly detrimental to young, symbiotic aphids; (2) dietary riboflavin is essential to aposymbiotic aphids; (3) dietary riboflavin remarkably improves the performance of aposymbiotic aphids. These results strongly suggest that young, symbiotic aphids are provided with riboflavin by their endosymbionts, Buchnera.

Bartonella koehlerae is reported for the first time to be a human pathogen that causes culture-negative endocarditis. It is also shown that this species, isolated twice before from domestic cats, can be recovered as well from a stray cat population in Israel. This work follows a recent report of the same case in which the causative agent was misidentified as B. henselae, based on serology and PCR-restriction fragment length polymorphism (RFLP) analysis (A. Schattner, O. Zimhony, B. Avidor, and M. Gilad, Lancet 361:1786, 2003). B. koehlerae was identified in the valvular tissue of an endocarditis patient by DNA sequencing of the PCR products of two Bartonella genes: the genes for citrate synthase (gltA) and riboflavin synthase (ribC). The commonly used PCR-RFLP analysis of the TaqI-digested gltA PCR product did not distinguish between B. koehlerae and B. quintana or between B. elizabethae and B. clarridgeiae. PmlI digestion of the gltA amplification product failed to differentiate between B. quintana, B. clarridgeiae, and B. elizabethae. RFLP analysis of the heat shock protein (htrA) gene by TaqI digestion misidentified B. koehlerae as B. henselae. However, RFLP analysis of the ribC PCR product, digested with TaqI, was able to distinguish between the human endocarditis-associated Bartonella species tested, B. henselae, B. quintana, B. elizabethae, and B. koehlerae, as well as between the cat-associated Bartonella species, B. henselae and B. clarridgeiae. Given the expanding number of Bartonella species emerging as human pathogens, it is suggested that PCR-RFLP analysis for the diagnosis of Bartonella infections target several genes and be coupled with DNA sequencing to avoid species identification.

B vitamins are some of the most commonly required biochemical cofactors in living systems. Therefore, cellular metabolism of marine vitamin-requiring (auxotrophic) phytoplankton and bacteria would likely be significantly compromised if B vitamins (thiamin B(1), riboflavin B(2), pyridoxine B(6), biotin B(7), and cobalamin B(12)) were unavailable. However, the factors controlling the synthesis, ambient concentrations, and uptake of these key organic compounds in the marine environment are still not well understood. Here, we report vertical distributions of five B vitamins (and the amino acid methionine) measured simultaneously along a latitudinal gradient through the contrasting oceanographic regimes of the southern California-Baja California coast in the Northeast Pacific margin. Although vitamin concentrations ranged from below the detection limits of our technique to 30 pM for B(2) and B(12) and to ∼500 pM for B(1), B(6), and B(7), each vitamin showed a different geographical and depth distribution. Vitamin concentrations were independent of each other and of inorganic nutrient levels, enriched primarily in the upper mesopelagic zone (depth of 100-300 m), and associated with water mass origin. Moreover, vitamin levels were below our detection limits (ranging from ≤0.18 pM for B(12) to ≤0.81 pM for B(1)) in extensive areas (100s of kilometers) of the coastal ocean, and thus may exert important constraints on the taxonomic composition of phytoplankton communities, and potentially also on rates of primary production and carbon sequestration.

BACKGROUND

A nutrient-wide approach may be useful to comprehensively test and validate associations between nutrients (derived from foods and supplements) and blood pressure (BP) in an unbiased manner.

RESULTS

Data from 4680 participants aged 40 to 59 years in the cross-sectional International Study of Macro/Micronutrients and Blood Pressure (INTERMAP) were stratified randomly into training and testing sets. US National Health and Nutrition Examination Survey (NHANES) four cross-sectional cohorts (1999-2000, 2001-2002, 2003-2004, 2005-2006) were used for external validation. We performed multiple linear regression analyses associating each of 82 nutrients and 3 urine electrolytes with systolic and diastolic BP in the INTERMAP training set. Significant findings were validated in the INTERMAP testing set and further in the NHANES cohorts (false discovery rate <5% in training, P<0.05 for internal and external validation). Among the validated nutrients, alcohol and urinary sodium-to-potassium ratio were directly associated with systolic BP, and dietary phosphorus, magnesium, iron, thiamin, folacin, and riboflavin were inversely associated with systolic BP. In addition, dietary folacin and riboflavin were inversely associated with diastolic BP. The absolute effect sizes in the validation data (NHANES) ranged from 0.97 mm Hg lower systolic BP (phosphorus) to 0.39 mm Hg lower systolic BP (thiamin) per 1-SD difference in nutrient variable. Inclusion of nutrient intake from supplements in addition to foods gave similar results for some nutrients, though it attenuated the associations of folacin, thiamin, and riboflavin intake with BP.

CONCLUSIONS

We identified significant inverse associations between B vitamins and BP, relationships hitherto poorly investigated. Our analyses represent a systematic unbiased approach to the evaluation and validation of nutrient-BP associations.

Riboflavin (vitamin B(2)) is the direct precursor of the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide, essential components of cellular biochemistry. In this work we investigated the unrelated proteins YpaA from Bacillus subtilis and PnuX from Corynebacterium glutamicum for a role in riboflavin uptake. Based on the regulation of the corresponding genes by a riboswitch mechanism, both proteins have been predicted to be involved in flavin metabolism. Moreover, their primary structures suggested that these proteins integrate into the cytoplasmic membrane. We provide experimental evidence that YpaA is a plasma membrane protein with five transmembrane domains and a cytoplasmic C terminus. In B. subtilis, riboflavin uptake was increased when ypaA was overexpressed and abolished when ypaA was deleted. Riboflavin uptake activity and the abundance of the YpaA protein were also increased when riboflavin auxotrophic mutants were grown in limiting amounts of riboflavin. YpaA-mediated riboflavin uptake was sensitive to protonophors and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation. In addition, we demonstrate that PnuX from C. glutamicum also is a riboflavin transporter. Transport by PnuX was not energy dependent and had high apparent affinity for riboflavin (K(m) 11 microM). Roseoflavin, a toxic riboflavin analog, appears to be a substrate of PnuX and YpaA. We propose to designate the gene names ribU for ypaA and ribM for pnuX to reflect that the encoded proteins function in riboflavin uptake and that the genes have different phylogenetic origins.