OBJECTIVE

To assess qualitative corneal changes and penetration of pulsed and continuous light accelerated crosslinking by in vivo confocal microscopy and corneal OCT.

METHODS

A total of 20 patients affected from progressive keratoconus were enrolled in the study. Ten eyes of 10 patients underwent an epithelium-off pulsed-light accelerated corneal collagen crosslinking (PL-ACXL) by the KXL UV-A source (Avedro Inc.) with 8 min (1 s on/1 s off) of UV-A exposure at 30 mW/cm(2) and energy dose of 7.2 J/cm(2); 10 eyes of 10 patients underwent an epithelium-off continuous-light accelerated corneal collagen crosslinking (CL-ACXL) at 30 mW/cm(2) for 4 min. Riboflavin 0.1% dextran-free plus hydroxyl-propyl-methylcellulose solution (VibeX Rapid, Avedro Inc.) was used for a 10-min corneal soaking. Treated eyes were examined by in vivo scanning laser confocal analysis and spectral anterior segment OCT at 1, 3, and 6 months.

RESULTS

Epithelial stratification and nerves regeneration improved in time, being complete at month 6 in both groups without endothelial damage. Keratocyte apoptosis in PL-ACXL was estimated at a mean depth of ∼200 μm, whereas an uneven demarcation line was detectable by confocal microscopy at a mean depth of 160 μm in CL-ACXL.

CONCLUSIONS

In vivo confocal microscopy and corneal OCT allowed a precise qualitative analysis of the cornea after epithelium-off PL-ACXL and CL-ACXL treatments. Apoptotic effect was higher in pulsed than in continuous light treatments, exceeding 200 μm in corneal stroma. According to different morphological data, the clinical efficacy of ACXL needs to be determined in a long-term follow-up and large cohort of patients.

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.

As part of an exploratory study of nutrition and senile cataract relationships between biochemical markers of nutritional status and senile cataract were examined in 112 subjects aged 40-70 y. Seventy-seven subjects had a cataract in at least one lens. Blood levels were determined for total carotenoids, vitamin A, vitamin D, vitamin E, vitamin C, riboflavin, thiamin, vitamin B-6, zinc, copper, selenium, and magnesium. Subjects were grouped into quintiles for each nutrient. Logistic regression was used to estimate the odds ratios (ORs) for cataract among subjects in the highest quintile and the middle three quintiles relative to subjects in the lowest quintile. ORs were adjusted for age, sex, race, and presence of diabetes. Results suggest that risk of cortical cataract was reduced for subjects in the highest quintile of vitamin D and total carotenoids and that persons with cataract may have lower levels of vitamin C and higher levels of vitamin B-6 and Se.

OBJECTIVE

To ascertain the efficacy of idebenone and multivitamin treatment in Leber's hereditary optic neuropathy (LHON).

METHODS

Two patients diagnosed of unilateral LHON were treated with megadoses of idebenone, vitamin C and riboflavin for one year. They were examined clinically before, during and after treatment.

RESULTS

No improvement of visual function was observed. Despite the idebenone treatment, in both cases the second eye became involved.

CONCLUSIONS

Despite previous reports of visual recovery with idebenone in patients with LHON, our experience shows that an effective treatment for Leber's disease remains to be found.

Several cross-sectional studies have focused on the low blood folate levels of depressed patients. However, no published studies have examined the association between dietary folate and current symptoms of depression in a general population. We investigated the association between dietary folate, cobalamin, pyridoxine and riboflavin and current symptoms of depression in a cross-sectional general population study. We recruited 2682 men aged between 42 and 60 y from eastern Finland. Those who had a previous history of psychiatric disorder were excluded (n = 146, 5.6% of the cohort). Depressive symptoms were assessed with the 18-item Human Population Laboratory Depression Scale. Those who scored 5 or more at baseline were considered to have elevated depressive symptoms (n = 228, 9.3% of the cohort). The participants were grouped into thirds according to their dietary folate intake. Those in the lowest third of energy-adjusted folate intake had a higher risk of being depressed [odds ratio (OR) 1.67, 95% CI = 1.19-2.35, P = 0.003] than those in the highest folate intake third. This increased risk remained significant after adjustment for smoking habits, alcohol consumption, appetite, BMI, marital status, education, adulthood socioeconomic status and total fat consumption (OR = 1.46, 95% CI = 1.01-2.12, P = 0.044). There were no associations between the intake of cobalamin, pyridoxine or riboflavin, and depression. These results indicate that nutrition may have a role in the prevention of depression.

OBJECTIVE

Scleral crosslinking by the photosensitizer riboflavin and ultraviolet A (UVA) has been shown to increase significantly the scleral biomechanical rigidity and might therefore become a possible sclera-based treatment modality for progressive myopia. In the present study, the long-term effect of the new crosslinking method on biomechanical properties was investigated in the rabbit sclera.

METHODS

A 10 x 10 mm sector of the equatorial sclera of nine Chinchilla rabbit eyes was treated in vivo using a UVA double diode of 370 nm with a surface irradiance of 3 mW/cm(2) and application of 0.1% riboflavin-5-phosphate drops as photosensitizer for 30 min. Three days, 4 months and 8 months postoperatively, biomechanical stress-strain measurements of the treated scleral strips were performed and compared to contralateral control sclera using a microcomputer-controlled biomaterial tester. In addition, routine histological controls were performed.

RESULTS

Following the crosslinking treatment, Young's modulus was increased by 320% after 3 days, 277% after 4 months and 502% after 8 months, and ultimate stress by 341% after 3 days, 131% after 4 months and 213.8% after 8 months versus the controls. The decrease in ultimate strain was between 24% and 44.8%. On histology, no tissue damage was detected.

CONCLUSIONS

Our new method of scleral collagen crosslinking proved very effective and constant over a time interval of up to 8 months in increasing the scleral biomechanical strength. Therefore, the new treatment might become an option for strengthening scleral tissue in progressive myopia and other conditions associated with weakened sclera. There were no side-effects on the retina or retinal pigment epithelium. The new crosslinking treatment could now be tested in a suitable myopia model (like the tree shrew) and finally in human eyes.

Complex II deficiency is a rare cause of mitochondrial respiratory chain defects with a prevalence of 2-23%. It is exclusively nuclear encoded and functions in the citric acid cycle by oxidizing succinate to fumarate and in the mitochondrial electron transport chain (ETC) by transferring electrons to ubiquinone. Of the four subunits, SDHA and SDHB are catalytic and SDHC and SDHD are anchoring. Mutations in SDHA and SDHAF1 (assembly factor) have been found in patients with CII deficiency and a mitochondrial phenotype. We present a patient with CII deficiency with a previously undescribed phenotype of dilated cardiomyopathy, left ventricular noncompaction, failure to thrive, hypotonia, and developmental delay. Also, a comprehensive review of 36 cases published in the literature was undertaken. The results show that CII deficiency has a variable phenotype with no correlation with residual complex activity in muscle although the phenotype and enzyme activities are comparable within a family. For some, the condition was fatal in infancy, others had multisystem involvement and some had onset in adulthood with mild symptoms and normal cognition. Neurological involvement is most commonly observed and brain imaging commonly shows leukoencephalopathy, Leigh syndrome, or cerebellar atrophy. Mutations in SDHAF1 are associated with leukoencephalopathy. Other organ systems like heart, muscle, and eyes are only involved in about 50% of the cases but cardiomyopathy is associated with high mortality and morbidity. In some patients, riboflavin has provided clinical improvement.

The abnormal metabolites-adipic, suberic, and sebacic acids-were detected in large amounts in the urine of a boy during a Reye's syndrome-like crisis. Substantial amounts of 5-OH-caproic acid, caproylglycine, glutaric acid, and 3-OH-butyric acid and moderately elevated amounts of ethylmalonic acid, methylsuccinic acid, 3-OH-isovaleric acid, and isovalerylglycine were also found. These metabolites were consistently present in urine samples collected in the boy's habitual condition after the attack. 1-[14C]-Palmitic acid was oxidized at a normal rate, whereas U-[14C]-Palmitic acid was oxidized at a reduced rate in cultured skin fibroblasts from the patient, thus indicating a defect at the level of medium- and/or short-chain fatty acid oxidation. Riboflavin medication (100 mg three times a day) significantly reduced the excreted amounts of pathologic metabolites, suggesting a flavineadeninedinucleotide-related acyl-CoA dehydrogenation defect as the cause of the disease. Carnitine in plasma was low in the patient (6 mumole/liter, controls 26-74 mumole/liter), suggesting carnitine deficiency as a secondary effect of the acyl-CoA dehydrogenation deficiency. The present patient, who presented with a Reye's syndrome-like attack, suffers from impaired dehydrogenation of acyl-CoA resulting in accumulation of acyl-CoA in the cells. Attacks with similar symptoms are seen in other acyl-CoA dehydrogenation deficiencies, such as glutaric aciduria types I and II, other types of C6-C10-dicarboxylic acidurias and isovaleric acidemia. Reduced flow through the acyl-CoA dehydrogenation steps may therefore be an ethiologic factor in Reye's syndrome. Several of the accumulated acyl-CoA's are toxic and may be responsible for some of the symptoms. The low carnitine level in plasma and the elevated esterified carnitine excretion in the present patient indicate that acyl-CoA accumulation may cause a functional carnitine deficiency by sequestration of carnitine as acyl-carnitines. As the inborn defect, systemic carnitine deficiency may exhibit symptoms like those of Reye's syndrome, it may be speculated whether functional carnitine deficiency in patients with accumulated acyl-CoA is another causal factor in the development of the symptoms during attacks.

Collagen gels have many favorable attributes for tissue engineering, but the gels undergo dramatic contraction when cells are added because of the weak noncovalent bonds that form during spontaneous gelation. We hypothesized that photochemically cross-linking collagen gels would make suitable scaffolds for tissue engineering with favorable cell viability and minimal gel contraction. Rose Bengal and riboflavin were chosen as candidate photo-initiators for gel cross-linking using 532- and 458-nm-light wavelengths, respectively. Chondrocyte viability was measured after initial gelation for several concentrations of initiators. Cell viability and gel contraction were then measured using chondrocytes and fibroblasts over 7 days of culture. Rose Bengal used at concentrations necessary for gelation resulted in little or no cell viability. Short-term viability results showed that 0.25- or 0.5-mM concentrations of riboflavin, and 40 s of illumination permitted more than 90% cell viability. Using riboflavin concentrations of 0.25 or 0.5 mM, long-term chondrocyte viability was 113.1 +/- 11.6% and 25.4 +/- 2.7%, respectively, at day 7. Although non-cross-linked chondrocyte constructs contracted to 59.9 +/- 11.8% of their original diameter and fibroblasts contracted to 24.9 +/- 5.0% of their original diameter by day 7, the cross-linked constructs retained 88.8 +/- 7.4% and 85.5 +/- 5.0% of the original diameter, respectively. In conclusion, by photochemically cross-linking collagen gels using riboflavin and visible light, stable gel scaffolds with favorable cell survival can be produced.

Mitochondrial complex I deficiency is the most common oxidative phosphorylation defect. Mutations have been detected in mitochondrial and nuclear genes, but the genetics of many patients remain unresolved and new genes are probably involved. In a consanguineous family, patients presented easy fatigability, exercise intolerance and lactic acidosis in blood from early childhood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity was improved by a supplement of riboflavin at high dosage. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594 C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to the loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild-type and mutant ACAD9. Wild-type, but not mutant, ACAD9 restored complex I activity. An unrelated patient with the same phenotype was compound heterozygous for c.380 G>A and c.1405 C>T, changing arginine-127 into glutamine and arginine-469 into tryptophan, respectively. These amino acids were highly conserved and the substitutions were not present in controls, making them very probably pathogenic. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment.

Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH(2))-utilizing monooxygenase, and FADH(2) was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH(2) and retarded its rapid oxidation by O(2). A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH(2)-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.

Injection drug use is an important factor in the spread of HIV infection, and strategies to enhance adherence to HIV therapeutics are critically important to controlling viral transmission and improving clinical outcomes. To this end, the authors sought (1) to enhance adherence to highly active antiretroviral therapy (HAART) among methadone-maintained injection drug users (IDUs) using modified directly observed therapy (MDOT), and (2) to define interactions between methadone and HAART and the potential contribution of drug interactions to adherence and HIV outcomes in this population. Adherence was explored here through a pilot, unblinded, 24-week study in a methadone maintenance program in which simplified HAART (efavirenz and didanosine [one daily] and a second nucleoside [twice daily]) was administered 6 days/week by clinic staff to HIV-infected IDUs (n = 5) with their methadone. Evening doses of riboflavin-tagged nucleoside and one full day of medication weekly were given as take home doses. As a result of HAART administration, four of five participants with mean viral load at baseline of 10(5) copies/ml had undetectable viral load by 8 weeks of treatment (p = .043). Methadone area under the curve (AUC) decreased by 55% (p = .007) within 2 weeks of initiating this HAART regimen, and a mean methadone dose increase of 52% was required. The authors conclude that MDOT is a promising intervention for the treatment of IDUs with HIV disease, though significant drug interactions must be monitored for carefully and rapidly addressed.

BACKGROUND

Naltrexone treatment of alcohol dependence is associated with adverse events that may limit its effectiveness. Consequently, understanding the impact of adverse events on medication compliance and treatment retention may enhance naltrexone therapy of alcoholism.

OBJECTIVE

To examine the relations among adverse events, drinking behavior, medication compliance and study retention in alcoholics receiving naltrexone for relapse prevention.

METHODS

The current report is based on analysis of data from 92 subjects who participated in two previously published studies. Moderate or severe adverse effects were monitored weekly and categorized as either neuropsychiatric (NP) or gastrointestinal (GI). Medication compliance was determined by weekly urinary riboflavin testing. Study retention was determined by the proportion of study weeks completed by the subject. The causal relations among adverse events, medication compliance and study retention were analyzed separately for NP and GI adverse events using regression-based recursive path models.

RESULTS

Both the NP and GI models fit the data well [NP model: chi 2(4) = 0.59, P = 0.96; GI model: chi 2(4) = 2.81, P = 0.59]. NP adverse events exerted little influence on medication compliance (beta = -0.17, P = 0.071), but directly decreased the length of study retention (beta = -0.35, P < 0.001). In contrast, there was a significant impact of GI adverse events on medication compliance (beta = -0.29, P = 0.002), but not directly on study retention (beta = -0.14, P = 0.081).

CONCLUSIONS

Future studies aimed at enhancing the effectiveness of naltrexone should examine ways of reducing both NP and GI adverse events, in order to enhance both medication compliance and treatment retention.

Glutaric acidemia type 1 (GA1) is overrepresented in the aboriginal population of Island Lake, Manitoba, and northwestern Ontario who speak the Ojibway-Cree (Oji-Cree) dialect. The carrier frequency in these communities has been predicted to be as high as 1 in 10 individuals. Prior to beginning newborn screening for GA1 in May 1998, 18 of 20 affected patients diagnosed at this center have been from these high-risk communities. Most have followed an acute encephalopathic course with permanent neurologic sequelae and high mortality. They excrete small amounts of glutaric acid and 3-hydroxyglutaric acid and have significant residual enzyme activity. A single homozygous mutation in glutaryl-CoA-dehydrogenase (GCDH IVS-1 + 5g right arrow t) has been identified in this population. DNA-based newborn screening targeted to our high-risk communities was begun in order to provide presymptomatic detection and treatment of affected patients. Of the first 1176 newborns screened, 4 affected infants were identified and treated with a low-protein diet, carnitine, and riboflavin. All 4 infants have required numerous hospitalizations for treatment of intercurrent illnesses. Eventually, 3 infants presented with acute dystonic encephalopathy and seizures along with permanent neurological sequelae. One of these infants died unexpectedly at home at 18 months of age. The fourth, now 9 months old, has had a gastrostomy tube placed to facilitate fluid replacement in addition to a standard treatment protocol and is doing well. The reasons for our initial disappointing outcomes in the first 3 of 4 affected babies are likely multiple. Based on our early experience and that of other centers screening newborns for GA1, current therapeutic strategies may be insufficient in preventing the occurrence of neurologic sequelae in some children. An incomplete understanding of the neurotoxic mechanisms underlying this devastating disorder hampers effective management.

Chronic alcohol consumption is a major risk factor for cancer of upper aero-digestive tract (oro-pharynx, hypopharynx, larynx and oesophagus), the liver, the colo-rectum and the breast. Evidence has accumulated that acetaldehyde is predominantly responsible for alcohol-associated carcinogenesis. Acetaldehyde is carcinogenic and mutagenic, binds to DNA and protein, destroys the folate molecule and results in secondary cellular hyper-regeneration. Acetaldehyde is produced by mucosal and cellular alcohol dehydrogenase, cytochrome P450 2E1 and through bacterial oxidation. Its generation and/or its metabolism is modulated as a result of polymorphisms or mutations of the genes responsible for these enzymes. Acetaldehyde can also be produced by oral bacteria. Smoking, which changes the oral bacterial flora, also increases salivary acetaldehyde. Cigarette smoke and some alcoholic beverages, such as Calvados, contain acetaldehyde. In addition, chronic alcohol consumption induces cytochrome P450 2E1 enxyme activity in mucosal cells, resulting in an increased generation of reactive oxygen species and in an increased activation of various dietary and environmental carcinogens. Deficiencies of riboflavin, Zn, folate and possibly retinoic acid may further enhance alcohol-associated carcinogenesis. Finally, methyl deficiency as a result of multiple alcohol-induced changes leads to DNA hypomethylation. A depletion of lipotropes, including methionine, choline, betaine and S-adenosylmethionine, as well as folate, results in the hypomethylation of oncogenes and may lead to DNA strand breaks, all of which are associated with increased carcinogenesis.

OBJECTIVE

To evaluate the safety and efficacy of staged ultraviolet A (UVA) cross-linking following intrastromal 0.1% riboflavin administration in eyes with advanced corneal edema.

METHODS

Ten eye bank corneas divided in two groups (n = 5) were placed on a pressurized artificial anterior chamber following Descemet's membrane stripping. Two consecutive corneal pockets (350- and 150-microm depth) were sequentially created using a femtosecond laser. Sequential intrastromal injections of 0.1% riboflavin (0.2 mL) followed by either UVA irradiation (15 mW/cm2) for 7 minutes or exposure to air were performed for each pocket. Corneal clarity and central thickness were measured before and after the two UVA cross-linking steps. The same steps were clinically applied in an 84-year-old woman with bullous keratopathy prior to corneal transplantation and followed for 6 months.

RESULTS

The corneal clarity improved in the treated but not the control eyes. The mean central corneal thickness was significantly reduced by 256 microm (ultrasound, P = .0002) and 273 miccrom (Scheimpflug, P = .0004) in treated eyes, but only 100 microm (ultrasound, P = .048) and 107 microm (Scheimpflug, P = .075) in the control eyes. The clinical treatment of corneal edema showed improved clarity and reduced central corneal thickness from 675 to 550 microm (ultrasound) and 696 to 571 microm (Scheimpflug) at 1 month. Best spectacle-corrected visual acuity improved from finger counting to 20/80 at 1 week and beyond, postponing corneal transplantation for>> 6 months.

CONCLUSIONS

Staged UVA cross-linking (15 mW/cm2) with femtosecond laser facilitated intrastromal 0.1% riboflavin administration may be a safe (no corneal scarring) and effective (marked reduction of edema) temporizing alternative method for managing bullous keratopathy.

The hydride carrier coenzyme F(420) contains the unusual chromophore 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Microbes that generate F(420) produce this FO moiety using a pyrimidine intermediate from riboflavin biosynthesis and the 4-hydroxyphenylpyruvate precursor of tyrosine. The fbiC gene, cloned from Mycobacterium smegmatis, encodes the bifunctional FO synthase. Expression of this protein in Escherichia coli caused the host cells to produce FO during growth, and activated cell-free extracts catalyze FO biosynthesis in vitro. FO synthase in the methanogenic euryarchaeon Methanocaldococcus jannaschii comprises two proteins encoded by cofG (MJ0446) and cofH (MJ1431). Both subunits were required for FO biosynthesis in vivo and in vitro. Cyanobacterial genomes encode homologs of both genes, which are used to produce the coenzyme for FO-dependent DNA photolyases. A molecular phylogeny of the paralogous cofG and cofH genes is consistent with the genes being vertically inherited within the euryarchaeal, cyanobacterial, and actinomycetal lineages. Ancestors of the cyanobacteria and actinomycetes must have acquired the two genes, which subsequently fused in actinomycetes. Both CofG and CofH have putative radical S-adenosylmethionine binding motifs, and pre-incubation with S-adenosylmethionine, Fe(2+), sulfide, and dithionite stimulates FO production. Therefore a radical reaction mechanism is proposed for the biosynthesis of FO.

ABSTRACT The role of riboflavin as an elicitor of systemic resistance and an activator of a novel signaling process in plants was demonstrated. Following treatment with riboflavin, Arabidopsis thaliana developed systemic resistance to Peronospora parasitica and Pseudomonas syringae pv. Tomato, and tobacco developed systemic resistance to Tobacco mosaic virus (TMV) and Alternaria alternata. Riboflavin, at concentrations necessary for resistance induction, did not cause cell death in plants or directly affect growth of the culturable pathogens. Riboflavin induced expression of pathogenesis-related (PR) genes in the plants, suggesting its ability to trigger a signal transduction pathway that leads to systemic resistance. Both the protein kinase inhibitor K252a and mutation in the NIM1/NPR1 gene which controls transcription of defense genes, impaired responsiveness to riboflavin. In contrast, riboflavin induced resistance and PR gene expression in NahG plants, which fail to accumulate salicylic acid (SA). Thus, riboflavin-induced resistance requires protein kinase signaling mechanisms and a functional NIM1/NPR1 gene, but not accumulation of SA. Riboflavin is an elicitor of systemic resistance, and it triggers resistance signal transduction in a distinct manner.

Cytochrome f from turnip and plastocyanin from French bean were noninvasively cross-linked in the presence of the carbodiimide EDC so that the exposed heme edge in the former protein abuts the acidic patch remote from the copper site in the latter [Morand, L.Z., Frame, M.K., Colvert, K.K., Johnson, D.A., Krogmann, D.W., & Davis, D.J. (1989) Biochemistry 28, 8039]. The molecular mass, reduction potentials, and UV-visible and ESR spectra of the covalent complex were consistent with the composition cyt/pc and with a lack of noticeable structural perturbations of the protein molecules. Isoelectric focusing showed the presence of N-acylurea groups, byproducts of the cross-linking reaction [Zhou, J.S., Brothers, H.M. II, Neddersen, J.P., Peerey, L.M., Cotton, T.M., & Kostić, N.M. (1992) Bioconjugate Chem. 3, 382]. Laser flash spectroscopy, with riboflavin semiquinone as the reductant, showed that the electrontransfer reaction within the covalent complex cyt(II)/pc(II) is either undetectably slow or reversible. The question was resolved by monitoring, during redox titrations, the 1H NMR line widths of the heme methyl groups in free ferricytochrome f and in this protein cross-linked to plastocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)

FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent K(m) and V(max) values for FMN equal to 1.3+/-0.7 microM and 4.4+/-1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 microM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria.

Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (pp) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.

The role of various micronutrients on the risk of renal cell cancer (RCC) was examined in a multicentric case-control study from Italy, in which information on dietary habits were collected using a validated food-frequency questionnaire. Cases were 767 patients (494 men and 273 women) with incident, histologically confirmed RCC; controls were 1,534 subjects (988 men and 546 women) admitted to the same hospitals as cases for a wide spectrum of acute, nonneoplastic conditions. After allowing for energy and other major covariates, a significant inverse association was found for vitamin E (odds ratio, OR, for the highest quintile of intake versus the lowest one 0.56, 95% confidence interval, CI 0.41-0.75), and vitamin C (OR = 0.72, 95% CI = 0.54-0.96), although the trend in risk for vitamin C was of borderline significance. No significant trend of decreasing risk was found for other micronutrients analyzed, although for most of them the risk estimates were below unity for intakes above the lowest. The ORs for the upper quintile of intake when compared with the lowest one were 0.80 (95% confidence interval, CI = 0.59-1.08) for retinol, 0.82 (95% CI = 0.61-1.10) for alpha-carotene, 0.90 (95% CI = 0.68-1.20) for beta-carotene, 0.94 (95% CI = 0.73-1.21) for beta-criptoxanthin, 0.85 (95% CI = 0.63-1.14) for lutein/zeaxanthin, 0.76 (95% CI = 0.57-1.01) for vitamin D, 0.75 (95% CI = 0.55-1.01) for thiamine, 0.88 (95% CI = 0.66-1.19) for riboflavin, 0.85 for vitamin B6 (95% CI = 0.64-1.13), 0.85 (95% CI = 0.64-1.12) for folate and 0.80 (95% CI = 0.60-1.07) for niacin. No meaningful associations emerged for lycopene (OR = 1.11). The present findings support a possible beneficial effect of vitamin E and C on RCC.

The desire to rid the blood supply of pathogens of all types has led to the development of many technologies aimed at the same goal--eradication of the pathogen(s) without harming the blood cells or generating toxic chemical agents. This is a very ambitious goal, and one that has yet to be achieved. One approach is to shun the 'one size fits all' concept and to target pathogen-reduction agents at the Individual component types. This permits the development of technologies that might be compatible with, for example, plasma products but that would be cytocidal and thus incompatible with platelet concentrates or red blood cell units. The technologies to be discussed include solvent detergent and methylene blue treatments--designed to inactivate plasma components and derivatives; psoralens (S-59--amotosalen) designed to pathogen-reduce units of platelets; and two products aimed at red blood cells, S-303 (a Frale--frangible anchor-linker effector compound) and Inactine (a binary ethyleneimine). A final pathogen-reduction material that might actually allow one material to inactivate all three blood components--riboflavin (vitamin B2)--is also under development. The sites of action of the amotosalen (S-59), the S-303 Frale, Inactine, and riboflavin are all localized in the nucleic acid part of the pathogen. Solvent detergent materials act by dissolving the plasma envelope, thus compromising the integrity of the pathogen membrane and rendering it non-infectious. By disrupting the pathogen's ability to replicate or survive, its infectivity is removed. The degree to which bacteria and viruses are affected by a particular pathogen-reducing technology relates to its Gram-positive or Gram-negative status, to the sporulation characteristics for bacteria, and the presence of lipid or protein envelopes for viruses. Concerns related to photoproducts and other breakdown products of these technologies remain, and the toxicology of pathogen-reduction treatments is a major ongoing area of investigation. Clearly, regulatory agencies have a major role to play in the evaluation of these new technologies. This chapter will cover the several types of pathogen-reduction systems, mechanisms of action, the inactivation efficacy for specific types of pathogens, toxicology of the various systems and the published research and clinical trial data supporting their potential usefulness. Due to the nature of the field, pathogen reduction is a work in progress and this review should be considered as a snapshot in time rather than a clear picture of what the future will bring.

Iron supplementation remains an important strategy for the prevention and treatment of iron deficiency anemia and can produce substantial improvements in the functional performance of iron deficient individuals and populations. Many potential benefits of iron supplementation require further exploration, including its effect on vitamin A and iodine metabolism. There is strong evidence that vitamin A and riboflavin deficiencies affect iron utilization from supplements and are important on a global scale, but little evidence that folate and vitamin B-12 deficiencies play a major causal role in the global burden of anemia. The efficacy of multiple micronutrient supplements for the prevention and treatment of anemia must be further evaluated. Because weekly supplementation with iron is effective at improving iron status, this option should be thoroughly explored and evaluated in the context of programs for the prevention and the treatment of iron deficiency and anemia. More conformation is warranted concerning the number of tablets that must be consumed in different situations, and the efficacy of supplying other micronutrients weekly with iron. Weekly supplementation programs may improve the logistical and economic constraints that currently limit the provision of supplements to the many target population groups for whom they are recommended, but usually fail to reach. Further work is required to clarify the purpose, delivery and outcomes of iron supplementation programs.