The filarial nematodes Brugia malayi, Wuchereria bancrofti and Onchocerca volvulus cause elephantiasis or dermatitis and blindness resulting in severe morbidity. Annually, 1.3 billion people are at risk of infection. Targeting the essential Wolbachia endobacteria of filarial nematodes with doxycycline has proven to be an effective therapy resulting in a block in embryogenesis, worm development and macrofilaricidal effects. However, doxycycline is contraindicated for a large portion of the at risk population. To identify new targets for anti-wolbachial therapy, understanding the molecular basis of the Wolbachia-filaria symbiosis is required. Using the B. malayi microarray we identified differentially expressed genes in the rodent filaria Litomosoides sigmodontis after depletion of Wolbachia which might have a role in symbiosis. The microarray data were filtered for regulated genes with a false discovery rate <5% and a>> or = 2-fold-change. Most of the genes were differentially expressed at day 36 of tetracycline treatment, when 99.8% of Wolbachia were depleted. Several classes of genes were affected, including genes for translation, transcription, folding/sorting of proteins, motility, structure and metabolic and signalling pathways. Quantitative PCR validated 60% of the genes found to be regulated in the microarray. A nuclear encoded heme-binding protein of the globin family was up-regulated upon loss of Wolbachia. Interestingly, mitochondrial encoded subunits of respiratory chain complexes containing heme and riboflavin were also up-regulated. No change in the expression of these genes was seen in tetracycline treated Wolbachia-free Acanthocheilonema viteae. As Wolbachia synthesise heme and filaria do not, we hypothesise that without the endosymbionts no functional heme-containing enzymes can be formed, leading to loss of energy metabolism which then results in up-regulation of the mitochondrial encoded subunits in an attempt to correct the deviation from homeostasis. Our results support targeting the Wolbachia heme synthesis pathway for the discovery of new anti-filarial drugs.

Normal microflora of the large intestine synthesize a number of water-soluble vitamins including riboflavin (RF). Recent studies have shown that colonic epithelial cells possess an efficient carrier-mediated mechanism for absorbing some of these micronutrients. The aim of the present study was to determine whether colonic cells also possess a carrier-mediated mechanism for RF uptake and, if so, to characterize this mechanism and study its cellular regulation. Confluent monolayers of the human-derived nontransformed colonic epithelial cells NCM460 and [(3)H]RF were used in the study. Uptake of RF was found to be 1) appreciable and temperature and energy dependent; 2) Na(+) independent; 3) saturable as a function of concentration with an apparent K(m) of 0.14 microM and V(max) of 3.29 pmol x mg protein(-1) x 3 min(-1); 4) inhibited by the structural analogs lumiflavin and lumichrome (K(i) of 1.8 and 14.1 microM, respectively) but not by the unrelated biotin; 5) inhibited in a competitive manner by the membrane transport inhibitor amiloride (K(i) = 0.86 mM) but not by furosemide, DIDS, or probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF uptake in RF-deficient and oversupplemented conditions, respectively; and 7) modulated by an intracellular Ca(2+)/calmodulin-mediated pathway. These studies demonstrate for the first time the existence of a specialized carrier-mediated mechanism for RF uptake in an in vitro cellular model system of human colonocytes. This mechanism appears to be regulated by extracellular substrate level and by an intracellular Ca(2+)/calmodulin-mediated pathway. It is suggested that the identified transport system may be involved in the absorption of bacterially synthesized RF in the large intestine and that this source of RF may contribute toward RF homeostasis, especially that of colonocytes.

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B(2)) to form FMN, an obligatory step in vitamin B(2) utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel beta barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3(10) helix, and a reverse turn followed by a short beta strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.

Particularly interesting cases of mutualistic endosymbioses come from the establishment of co-obligate associations of more than one species of endosymbiotic bacteria. Throughout symbiotic accommodation from a free-living bacterium, passing through a facultative stage and ending as an obligate intracellular one, the symbiont experiences massive genomic losses and phenotypic adjustments. Here, we scrutinized the changes in the coevolution of Serratia symbiotica and Buchnera aphidicola endosymbionts in aphids, paying particular attention to the transformations undergone by S. symbiotica to become an obligate endosymbiont. Although it is already known that S. symbiotica is facultative in Acyrthosiphon pisum, in Cinara cedri it has established a co-obligate endosymbiotic consortium along with B. aphidicola to fulfill the aphid's nutritional requirements. The state of this association in C. tujafilina, an aphid belonging to the same subfamily (Lachninae) that C. cedri, remained unknown. Here, we report the genome of S. symbiotica strain SCt-VLC from the aphid C. tujafilina. While being phylogenetically and genomically very closely related to the facultative endosymbiont S. symbiotica from the aphid A. pisum, it shows a variety of metabolic, genetic, and architectural features, which point toward this endosymbiont being one step closer to an obligate intracellular one. We also describe in depth the process of genome rearrangements suffered by S. symbiotica and the role mobile elements play in gene inactivations. Finally, we postulate the supply to the host of the essential riboflavin (vitamin B2) as key to the establishment of S. symbiotica as a co-obligate endosymbiont in the aphids belonging to the subfamily Lachninane.

OBJECTIVE

To investigate the influence of osmolarity on the transepithelial permeability of riboflavin solutions in a cross-linking procedure.

METHODS

Several riboflavin 0.1% solutions that contained different NaCl and benzalkonium chloride (BAC) concentrations were applied to 36 rabbit eyes for 30 min. To serve as a control, the epithelium was removed in group A (standard protocol). The groups then received the following solutions: (A) riboflavin 0.1% in NaCl 0.9% solution; (B) riboflavin 0.1% in NaCl 0.44% solution with BAC 0.02%; (C) riboflavin 0.1% in NaCl 0.44% solution with BAC 0.01%; (D) riboflavin 0.1% in NaCl 0.44% solution without BAC; (E) riboflavin 0.1% in NaCl 0.9% solution with BAC 0.02%; and (F) riboflavin 0.1% in NaCl 0.9% solution without BAC. Six eyes in each group were treated. The absorption coefficients of the corneas were measured to characterize the riboflavin penetration into the cornea.

RESULTS

There is a large difference in the transepithelial riboflavin penetration of riboflavin 0.1% + BAC 0.02% solutions that contain different NaCl concentrations (NaCl 0.9% versus NaCl 0.44%). The absorption coefficients differed by more than a factor of two (P = 0.004). No statistically significant difference was found between riboflavin 0.1% in NaCl 0.44% solution containing BAC 0.02% and BAC 0.01%. Compared to the standard protocol, these solutions resulted in an absorption coefficient of 37% (BAC 0.02%) and 33% (BAC 0.01%) of the standard epithelium-off procedure.

CONCLUSIONS

The transepithelial riboflavin solution should contain no dextran, but it should include 0.01% BAC and 0.44% NaCl to promote the permeability of riboflavin through the epithelium, resulting in a sufficient concentration of riboflavin in the corneal stroma.

The lumazine synthase/riboflavin synthase of Bacillus subtilis is a bifunctional enzyme complex catalysing the formation of riboflavin from 5-amino-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and L-3,4-dihydroxy-2-butanone-4-phosphate via 6,7-dimethyl-8-ribityllumazine. The complex is composed of 3 alpha (riboflavin synthase) subunits and 60 beta (lumazine synthase) subunits and has a relative mass of 1 MDa. The 60 beta subunits are arranged in an icosahedral capsid enclosing the alpha trimer in the central core. The protein was crystallised, and an X-ray structure of the icosahedral capsid was obtained at 3.3 A resolution with a crystallographic R-factor of 0.33. Hollow, icosahedral capsids consisting of 60 beta subunits can be obtained by inhibitor-driven renaturation of isolated beta subunits. They catalyse the formation of 6,7-dimethyl-8-ribityllumazine at the same rate as the native alpha 3 beta 60 complex and can be crystallised in two different hexagonal and one monoclinic form. Crystallographic intensity data of the monoclinic crystals to a resolution of 2.4 A were obtained using synchrotron radiation and an image plate detector system. The orientation of the icosahedral molecules in the monoclinic cell was deduced by real space vector search procedures from a 3.5 A Patterson map. Phases were calculated from the model of the alpha 3 beta 60 protein and were extended by cyclic averaging exploring the 30-fold redundancy of the electron density. The 2.4 A map allowed us to refine the existing atomic model of lumazine synthase. The refined model includes 154 amino acid residues, one inhibitor molecule, 58 water molecules and one phosphate ion. Applying non-crystallographic-symmetry restraints the crystallographic R-factor is 16.7% for 100,092 reflections between 10 and 2.4 A. The chain folding of the beta subunits is closely similar to the native alpha 3 beta 60 enzyme. The lumazine synthase bears resemblance to the sugar binding proteins. The significantly higher resolution compared to the alpha 3 beta 60 structure determination allows a detailed description of the substrate analogue binding site. The environment of the 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione inhibitor is particularly rigid, and the chain segments involved in forming the active site are highly conserved for lumazine synthases of different species. A residual density feature in the final map is interpreted as a bound phosphate which mimics the binding of the second substrate. We discuss the reaction mechanism on this structural basis.

Riboflavin-responsive forms of multiple acyl-CoA dehydrogenation deficiency (RR-MADD) have been known for years, but with presumed defects in the formation of the flavin adenine dinucleotide (FAD) co-factor rather than genetic defects of electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). It was only recently established that a number of RR-MADD patients carry genetic defects in ETF-QO and that the well-documented clinical efficacy of riboflavin treatment may be based on a chaperone effect that can compensate for inherited folding defects of ETF-QO. In the present study, we investigate the molecular mechanisms and the genotype-phenotype relationships for the riboflavin responsiveness in MADD, using a human HEK-293 cell expression system. We studied the influence of riboflavin and temperature on the steady-state level and the activity of variant ETF-QO proteins identified in patients with RR-MADD, or non- and partially responsive MADD. Our results showed that variant ETF-QO proteins associated with non- and partially responsive MADD caused severe misfolding of ETF-QO variant proteins when cultured in media with supplemented concentrations of riboflavin. In contrast, variant ETF-QO proteins associated with RR-MADD caused milder folding defects when cultured at the same conditions. Decreased thermal stability of the variants showed that FAD does not completely correct the structural defects induced by the variation. This may cause leakage of electrons and increased reactive oxygen species, as reflected by increased amounts of cellular peroxide production in HEK-293 cells expressing the variant ETF-QO proteins. Finally, we found indications of prolonged association of variant ETF-QO protein with the Hsp60 chaperonin in the mitochondrial matrix, supporting indications of folding defects in the variant ETF-QO proteins.

OBJECTIVE

To compare nutrient intakes between vegetarians and nonvegetarians with similar health practices, and to assess relationships with eating behavior scores from the Three-Factor Eating Questionnaire.

METHODS

Survey.

METHODS

Metropolitan area in western Canada.

METHODS

Subjects (n = 45) were participants in a study comparing subclinical menstrual disturbances between vegetarians and nonvegetarians. To be included, women had to be 20 to 40 years old, be weight stable with a body mass index (BMI; kg/m2) of 18 to 25, be a nonsmoker, exercise 7 hours a week or less, consume one alcoholic drink or less a day, and not be using oral contraceptives. Nonvegetarians (n = 22) ate red meat three times a week or more, and vegetarians (n = 23, 8 vegans and 15 lactovegetarians) had excluded all meat, fish, and poultry for 2 years or more.

METHODS

Nutrient intake assessed by three 3-day diet records; supplement use; body composition; and dietary restraint (conscious limitation of food intake), disinhibition, and hunger assessed by the Three-Factor Eating Questionnaire.

METHODS

Anthropometric variables, nutrient intakes, and eating behavior scores were compared between vegetarians and nonvegetarians using unpaired t tests, and among vegans, lactovegetarians, and nonvegetarians using one-way analysis of variance and Duncan's test. Supplement use was compared using chi 2 analysis. The Pearson correlation coefficient was used to evaluate relationships between variables.

RESULTS

Diets of all women adhered closely to current nutrition recommendations. Vegetarians had lower protein and cholesterol intakes and higher percentage of energy as carbohydrate, ratio of polyunsaturated fat to saturated fat (P:S ratio), and fiber intake than nonvegetarians. Vegetarians had lower riboflavin, niacin, vitamin B-12, zinc, and sodium intakes and higher folate, vitamin C, and copper intakes. However, many differences were not apparent between the subgroup of lactovegetarians and nonvegetarians (their P:S ratios and carbohydrate, fiber, riboflavin, folate, vitamin C, and copper intakes were similar). In contrast, differences existed between the lactovegetarian and the vegan subgroups. Supplement use was similar between groups, except for greater vitamin C use by vegetarians. Vegetarians were leaner than nonvegetarians, had lower restraint scores, and had significant associations between restraint and BMI (r = .49; P < .05) and energy per kilogram body weight (r = -.60; P < .01).

CONCLUSIONS

Current nutrition recommendations can be attained by vegetarians and nonvegetarians alike, but nutrient intakes cannot be inferred from dietary pattern. In this study, the intakes of health-conscious nonvegetarians and lactovegetarians were more similar than the intakes of lactovegetarians and vegans. Vegans' calcium and vitamin B-12 intakes may need attention. Vegetarians' lower restraint scores suggest that they are not at increased risk for eating disorders.

Hydrogen peroxide, lethal for human cells, is produced in Dulbecco's modified Eagle's tissue culture medium when exposed to "daylight" fluorescent light. Addition of pure H2O2 and use of the enzyme catalase demonstrate that about 40% of the toxicity in irradiated medium is due to generated peroxide. Riboflavin and tryptophan, or riboflavin and tyrosine, are the components necessary for formation of lethal levels of H2O2 during light exposure.

In Linxian China, the esophageal/gastric cardia cancer mortality rates are among the highest in the world. There is suspicion that the population's chronic deficiencies of multiple micronutrients are etiologically involved. We conducted two randomized, placebo-controlled nutrition intervention trials to test the effects of vitamin and mineral supplements in lowering the rates of esophageal/gastric cancer. In the first trial, the dysplasia trial, 3318 adults with a cytological diagnosis of esophageal dysplasia received daily supplementation with 26 vitamins and minerals in doses typically 2-3 times the United States Recommended Daily Allowances, or placebos, for 6 years. The second trial, the general population trial, involved 29,584 adults and used a one-half replicate of a 2(4) factorial experimental design which tested the effects of four combinations of nutrients: A, retinol and zinc; B, riboflavin and niacin; C, vitamin C and molybdenum; and D, beta-carotene, vitamin E, and selenium. Doses for these daily supplements ranged from 1 to 2 times the United States Recommended Daily Allowances, and the different vitamin/mineral combinations or placebos were taken for a period of 5.25 years. As part of the general population trial, and end-of-intervention endoscopy survey was carried out in a small (1.3%) sample of subjects to see if supplementation affected the prevalence of dysplasia and early cancer. Herein we review the methods of these trials and the results of the endoscopic survey. Fifteen esophageal and 16 gastric cancers were identified in endoscopic biopsies from the 391 subjects evaluated from two villages, and nearly all were asymptomatic. No significant reductions in the prevalence of esophageal or gastric dysplasia or cancer were seen with any of the four supplement groups. However, the prevalence of gastric cancer among participants receiving retinol and zinc was 62% lower than those not receiving those supplements (P = 0.09), while participants receiving beta-carotene, vitamin E, and selenium had a 42% reduction in esophageal cancer prevalence (0.34). We have reported separately that cancer mortality over the entire 5.25-year period was significantly reduced among those receiving beta-carotene, vitamin E, and selenium. The findings from the overall trial and the endoscopic sample offer a hopeful sign and should encourage additional studies with these agents in larger numbers of subjects.

Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

Malnutrition is a common feature of chronic liver diseases that is often associated with a poor prognosis including worsening of clinical outcome, neuropsychiatric complications as well as outcome following liver transplantation. Nutritional assessment in patients with cirrhosis is challenging owing to confounding factors related to liver failure. The objectives of nutritional intervention in cirrhotic patients are the support of liver regeneration, the prevention or correction of specific nutritional deficiencies and the prevention and/or treatment of the complications of liver disease per se and of liver transplantation. Nutritional recommendations target the optimal supply of adequate substrates related to requirements linked to energy, protein, carbohydrates, lipids, vitamins and minerals. Some issues relating to malnutrition in chronic liver disease remain to be addressed including the development of an appropriate well-validated nutritional assessment tool, the identification of mechanistic targets or therapy for sarcopenia, the development of nutritional recommendations for obese cirrhotic patients and liver-transplant recipients and the elucidation of the roles of vitamin A hepatotoxicity, as well as the impact of deficiencies in riboflavin and zinc on clinical outcomes. Early identification and treatment of malnutrition in chronic liver disease has the potential to lead to better disease outcome as well as prevention of the complications of chronic liver disease and improved transplant outcomes.

OBJECTIVE

Changes in in vitro platelet quality parameters during platelet storage are associated with a decrease of in vivo platelet viability after platelet transfusion. Many attempts have been made to identify the most predictable in vitro parameters for in vivo performance. We used a riboflavin-based ultraviolet (UV) light treatment process designed to inactivate pathogens and white blood cell (WBC) contaminants in blood products as a model system in which to study the correlation of in vitro cell quality with in vivo viability.

METHODS

Platelet products (n = 18) were collected by a standard Trima apheresis procedure and treated with one of three dose levels of UV light (0, 7.2 or 12.4 J/ml) in the presence of 50 microm riboflavin. Lactate production, glucose consumption and P-selectin expression, pH, pCO(2), pO(2), hypotonic shock response and swirl were measured during 5 days of platelet storage post-UV/RB treatment. Aliquots of these products were radiolabelled on day 5 of storage and were subsequently used to determine platelet recovery and survival time in autologous subjects.

RESULTS

The responses of in vitro cell quality were observed to occur in a UV dose-dependent manner. Lactate production and pH were identified as the parameters most strongly correlated with platelet in vivo recovery, which ranged from 5 to 82%. The correlation coefficients (r) for lactate production and pH with in vivo recovery in human subjects were 0.9090 and 0.8831 with P-values of 0.007 and 0.031, respectively. Lactate production and pH were also found to be correlated with platelet survival time, with correlation coefficients of 0.8063 and 0.8384 (the P values were 0.01 and 0.001, respectively).

CONCLUSIONS

Using conditions of riboflavin-based UV light treatment, lactate production and pH were identified as having the highest correlations with recovery and survival of radiolabelled platelets in healthy subjects.

In China, many species of edible wild-grown mushrooms are appreciated as food and also found use in traditional Chinese medicine. In this mini-review, for the first time, is summarized and discussed data available on chemical components of nutritional significance for wild-grown mushrooms collected from China. We aimed to update and discuss the latest data published on ash, fat, carbohydrates, fibre, proteins, essential amino acids and nonessential amino acids, some essential (P, K, Na, Ca, Mg, Fe, Mn, Zn, Cu) and toxic elements (As, Hg, Cd, Pb), vitamins (thiamine, riboflavin, niacin, tocopherol, vitamin D), flavour and taste compounds, antioxidants and also on less studied organic compounds (lectin, adustin, ribonuclease and nicotine) contents of wild-grown mushrooms.

BACKGROUND

Riboflavin seems to have a promising effect on migraine in adults. The present study examines whether riboflavin has a prophylactic effect on migraine in children.

OBJECTIVE

To investigate whether riboflavin in a dosage of 50 mg/day has a prophylactic effect on migraine attacks in young children.

METHODS

This randomised, placebo-controlled, double-blind, cross-over trial included 42 children (aged 6-13 years) with migraine of whom 14 children were also suffering from tension-type headache. Following a 4-week baseline period, all children received placebo for 16 weeks then riboflavin for 16 weeks (or vice versa) with a washout period of 4 weeks in between. The primary outcome measure was reduction in mean frequency of migraine attacks and tension-type headache in the last 4 weeks at the end of the riboflavin and placebo phase, compared with the preceding baseline or wash-out period. Secondary outcome measures were mean severity and mean duration of migraine and tension-type headaches in the last 4 weeks at the end of the riboflavin and placebo phase, compared with the preceding baseline or wash-out period.

RESULTS

No significant difference in the reduction of mean frequency of migraine attacks in the last month of treatment was found between placebo and riboflavin (P = 0.44). However, a significant difference in reduction of mean frequency of headaches with a tension-type phenotype was found in favour of the riboflavin treatment (P = 0.04).

CONCLUSIONS

In this group of children with migraine, there is no evidence that 50 mg riboflavin has a prophylactic effect on migraine attacks. We found some evidence that 50 mg riboflavin may have a prophylactic effect on interval headaches that may correspond to mild migraine attacks or tension-type headache attacks in children with migraine.

Pathophysiological mechanisms of cataract formation include deficient glutathione levels contributing to a faulty antioxidant defense system within the lens of the eye. Nutrients to increase glutathione levels and activity include lipoic acid, vitamins E and C, and selenium. Cataract patients also tend to be deficient in vitamin A and the carotenes, lutein and zeaxanthin. The B vitamin riboflavin appears to play an essential role as a precursor to flavin adenine dinucleotide (FAD), a co-factor for glutathione reductase activity. Other nutrients and botanicals, which may benefit cataract patients or help prevent cataracts, include pantethine, folic acid, melatonin, and bilberry. Diabetic cataracts are caused by an elevation of polyols within the lens of the eye catalyzed by the enzyme aldose reductase. Flavonoids, particularly quercetin and its derivatives, are potent inhibitors of aldose reductase. Glaucoma is characterized by increased intraocular pressure (IOP) in some but not all cases. Some patients with glaucoma have normal IOP but poor circulation, resulting in damage to the optic nerve. Faulty glycosaminoglycan (GAG) synthesis or breakdown in the trabecular meshwork associated with aqueous outflow has also been implicated. Similar to patients with cataracts, those with glaucoma typically have compromised antioxidant defense systems as well. Nutrients that can impact GAGs such as vitamin C and glucosamine sulfate may hold promise for glaucoma treatment. Vitamin C in high doses has been found to lower IOP via its osmotic effect. Other nutrients holding some potential benefit for glaucoma include lipoic acid, vitamin B12, magnesium, and melatonin. Botanicals may offer some therapeutic potential. Ginkgo biloba increases circulation to the optic nerve; forskolin (an extract from Coleus forskohlii) has been used successfully as a topical agent to lower IOP; and intramuscular injections of Salvia miltiorrhiza have shown benefit in improving visual acuity and peripheral vision in people with glaucoma.

There has lately been a renewed interest in Riboflavin owing to insight into its recognition as an essential component of cellular biochemistry. The knowledge of the mechanisms and regulation of intestinal absorption of riboflavin and its health implications has significantly been expanded in recent years. The purpose of this review is to provide an overview of the importance of riboflavin, its absorption and metabolism in health and diseased conditions, its deficiency and its association with various health diseases, and metabolic disorders. Efforts have been made to review the available information in literature on the relationship between riboflavin and various clinical abnormalities. The role of riboflavin has also been dealt in the prevention of a wide array of health diseases like migraine, anemia, cancer, hyperglycemia, hypertension, diabetes mellitus, and oxidative stress directly or indirectly. The riboflavin deficiency has profound effect on iron absorption, metabolism of tryptophan, mitochondrial dysfunction, gastrointestinal tract, brain dysfunction, and metabolism of other vitamins as well as is associated with skin disorders. Toxicological and photosensitizing properties of riboflavin make it suitable for biological use, such as virus inactivation, excellent photosensitizer, and promising adjuvant in chemo radiotherapy in cancer treatment. A number of recent studies have indicated and highlighted the cellular processes and biological effects associated with riboflavin supplementation in metabolic diseases. Overall, a deeper understanding of these emerging roles of riboflavin intake is essential to design better therapies for future.

BACKGROUND

Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α, and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1-/-, and peroxisome proliferator-activated receptor-α,-/- (PPAR-α,-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes.

RESULTS

There was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; P < 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α, secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α, secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α, secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1-/- and PPAR-α,-/- knockout mice. Results of gene expression studies for TNF-α, and iNOS were generally consistent with results obtained for TNF-α, protein and NO production observed with four strains of mice.

CONCLUSIONS

Results of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α, secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.

OBJECTIVE

This article reviews the evidence base for the preventive treatment of migraine.

RESULTS

Evidence-based guidelines for the preventive treatment of migraine have recently been published by the American Academy of Neurology (AAN) and the Canadian Headache Society (CHS), providing valuable guidance for clinicians. Strong evidence exists to support the use of metoprolol, timolol, propranolol, divalproex sodium, sodium valproate, and topiramate for migraine prevention, according to the AAN. Based on best available evidence, adverse event profile, and expert consensus, topiramate, propranolol, nadolol, metoprolol, amitriptyline, gabapentin, candesartan, Petasites (butterbur), riboflavin, coenzyme Q10, and magnesium citrate received a strong recommendation for use from the CHS.

CONCLUSIONS

Migraine preventive drug treatments are underutilized in clinical practice. Principles of preventive treatment are important to improve compliance, minimize side effects, and improve patient outcomes. Choice of preventive treatment of migraine should be based on the presence of comorbid and coexistent illness, patient preference, reproductive potential and planning, and best available evidence.

Cytoplasmic and mitochondrial tRNAs contain several pseudouridylation sites, and the tRNA:Psi-synthase acting at position 32 had not been identified in Saccharomyces cerevisiae. By combining genetic and biochemical analyses, we demonstrate that two enzymes, Rib2/Pus8p and Pus9p, are required for Psi32 formation in cytoplasmic and mitochondrial tRNAs, respectively. Pus9p acts mostly in mitochondria, and Rib2/Pus8p is strictly cytoplasmic. This is the first case reported so far of two distinct tRNA modification enzymes acting at the same position but present in two different compartments. This peculiarity may be the consequence of a gene fusion that occurred during yeast evolution. Indeed, Rib2/Pus8p displays two distinct catalytic activities involved in completely unrelated metabolism: its C-terminal domain has a DRAP-deaminase activity required for riboflavin biogenesis in the cytoplasm, whereas its N-terminal domain carries the tRNA:Psi32-synthase activity. Pus9p has only a tRNA:Psi32-synthase activity and contains a characteristic mitochondrial targeting sequence at its N terminus. These results are discussed in terms of RNA:Psi-synthase evolution.

The ribG gene at the 5' end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable.

OBJECTIVE

Corneal collagen cross-linking (CXL) by the use of riboflavin and ultraviolet-A light (UVA) is a promising and novel treatment for keratoconus and other ectatic disorders. Since CXL results in enhanced corneal stiffness, this study tested the hypothesis that CXL-induced stiffening would be proportional to the collagen autofluorescence intensity measured with nonlinear optical (NLO) microscopy.

METHODS

Rabbit eyes (n = 50) were separated into five groups including: (1) epithelium intact; (2) epithelium removed; (3) epithelium removed and soaked in riboflavin, (4) epithelium removed and soaked in riboflavin, with 15 minutes of UVA exposure; and (5) epithelium removed and soaked in riboflavin, with 30 minutes of UVA exposure. Corneal stiffness was quantified by measuring the force required to displace the cornea 500 μm. Corneas were then fixed in paraformaldehyde and sectioned, and the collagen autofluorescence over the 400- to 450-nm spectrum was recorded.

RESULTS

There was no significant difference in corneal stiffness among the three control groups. Corneal stiffness was significantly and dose dependently increased after UVA (P < 0.0005). Autofluorescence was detected only within the anterior stroma of the UVA-treated groups, with no significant difference in the depth of autofluorescence between different UVA exposure levels. The signal intensity was also significantly increased with longer UVA exposure (P < 0.001). Comparing corneal stiffness with autofluorescence intensity revealed a significant correlation between these values (R(2) = 0.654; P < 0.0001).

CONCLUSIONS

The results of this study indicate a significant correlation between corneal stiffening and the intensity of collagen autofluorescence after CXL. This finding suggests that the efficacy of CXL in patients could be monitored by assessing collagen autofluorescence.

Riboflavin (vitamin B2) is absorbed in the small intestine by the human riboflavin transporters RFVT1 and RFVT3. A third riboflavin transporter (RFVT2) is expressed in the brain. In 2010 it was demonstrated that mutations in the riboflavin transporter genes SLC52A2 (coding for RFVT2) and SLC52A3 (coding for RFVT3) cause a neurodegenerative disorder formerly known as Brown-Vialetto-Van Laere (BVVL) syndrome, now renamed to riboflavin transporter deficiency. Five years after the diagnosis of the first patient we performed a review of the literature to study the presentation, treatment and outcome of patients with a molecularly confirmed diagnosis of a riboflavin transporter deficiency.

A search was performed in Medline, Pubmed using the search terms 'Brown-Vialetto-Van Laere syndrome' and 'riboflavin transporter' and articles were screened for case reports of patients with a molecular diagnosis of a riboflavin transporter deficiency.

Reports on a total of 70 patients with a molecular diagnosis of a RFVT2 or RTVT3 deficiency were retrieved. The riboflavin transporter deficiencies present with weakness, cranial nerve deficits including hearing loss, sensory symptoms including sensory ataxia, feeding difficulties and respiratory difficulties which are caused by a sensorimotor axonal neuropathy and cranial neuropathy. Biochemical abnormalities may be absent and the diagnosis can only be made or rejected by molecular analysis of all genes. Treatment with oral supplementation of riboflavin is lifesaving. Therefore, if a riboflavin transporter deficiency is suspected, treatment must be started immediately without first awaiting the results of molecular diagnostics.

Bacterial nitroreductases are NAD(P)H-dependent flavoenzymes which catalyze the oxygen-insensitive reduction of nitroaromatics, quinones, and riboflavin derivatives. Despite their broad substrate specificity, their reactivity is very specific for two-electron, not one-electron, chemistry. We now describe the thermodynamic properties of the flavin mononucleotide cofactor of Enterobacter cloacae nitroreductase (NR), determined under a variety of solution conditions. The two-electron redox midpoint potential of NR is -190 mV at pH 7.0, and both the pH dependence of the midpoint potential and the optical spectrum of the reduced enzyme indicate that the transition is from neutral oxidized flavin to anionic flavin hydroquinone. The one-electron-reduced semiquinone states of both the free enzyme and an NR-substrate analogue complex are strongly suppressed based on optical spectroscopy and electron paramagnetic resonance measurements. This can explain the oxygen insensitivity of NR and its homologues, as it makes the execution of one-electron chemistry thermodynamically unfavorable. Therefore, we have established a chemical basis for the recent finding that a nitroreductase is a member of the soxRS oxidative defense regulon in Escherichia coli [Liochev, S. I., Hausladen, A., Fridovich, I. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (7), 3537-3539]. We also report binding affinities for the FMN cofactor in all three oxidation states either determined fluorometrically or calculated using thermodynamic cycles. Thus, we provide a detailed picture of the thermodynamics underlying the unusual activity of NR.