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Lysosome function is compromised during aging and in many disease states. Interventions that promote lysosomal activity and acidification are thus of prime interest as treatments for longevity and health. Intracellular pH can be controlled by the exchange of protons for inorganic ions, and in cells from microbes to man, when potassium is restricted in the growth medium, the cytoplasm becomes acidified. Here we use a yeast model to show that potassium limited-cells exhibit hallmarks of increased acidity in the vacuole, the analog of the lysosome, and live long by a mechanism that requires the vacuolar machinery. The emerging picture is one in which potassium restriction shores up vacuolar acidity and function, conferring health benefits early in life and extending viability into old age. Against the backdrop of well-studied protein and carbohydrate restrictions that extend lifespan and healthspan, our work establishes a novel pro-longevity paradigm of inorganic nutrient limitation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Increasing evidence suggests that lifestyle factors may decrease the risk of prostate cancer progression. Lifestyle guidelines and tools may support lifestyle modification after diagnosis., Objective: To determine the feasibility and acceptability of a digital lifestyle intervention among men with prostate cancer., Design, Setting, and Participants: A 12-wk pilot randomized controlled trial among 76 men with clinical stage T1-T3a prostate cancer. Eligibility included Internet access, no contraindications to aerobic exercise, and engaging in four or fewer of eight targeted behaviors at baseline., Intervention: Website, Fitbit One, and text messaging to facilitate adoption of eight behaviors: vigorous activity, smoking cessation, and six diet improvements., Outcome Measurements and Statistical Analysis: Our primary outcomes were feasibility and acceptability based on recruitment and user data, and surveys, respectively. Secondarily, we evaluated the change in eight lifestyle behaviors, and also objective physical activity. Each factor was assigned one point, for an overall "P8 score" (range 0-8). Analysis of covariance (ANCOVA) was conducted. Exploratory outcomes included quality of life, anthropometrics, and circulating biomarkers after 12wk, and behaviors after 1yr., Results and Limitations: At baseline, men in both arms met a median of three targeted behaviors. Sixty-four men (n=32 per arm) completed the study; 88% completed 12-wk assessments (intervention, 94%; control, 82%). Intervention participants wore their Fitbits a median of 82d (interquartile range [IQR]: 72-83), replied to a median of 71% of text messages (IQR: 57-89%), and visited the website a median of 3d (IQR: 2-5) over 12wk. Median (IQR) absolute changes in the P8 score from baseline to 12wk were 2 (1, 3) for the intervention and 0 (-1, 1) for the control arm. The estimated mean score of the intervention arm was 1.5 (95% confidence interval: 0.7, 2.3) higher than that of the control arm at 12wk (ANCOVA p<0.001). Changes were driven by diet rather than exercise. Limitations include self-reported diet and exercise data., Conclusions: Overall, in this novel pilot trial, the intervention was feasible and acceptable to men with prostate cancer. Next steps include improving the intervention to better meet individuals' needs and focusing on increasing physical activity in men not meeting nationally recommended physical activity levels., Patient Summary: Tailored print materials combined with technology integration, including the use of a website, text messaging, and physical activity trackers, helped men with prostate cancer adopt healthy lifestyle habits, in particular recommended dietary changes, in the Prostate 8 pilot trial., (Copyright © 2018. Published by Elsevier B.V.)

Background: Meningococcal epidemics in Sub-Sahara caused by serogroup A strains are controlled by a group A polysaccharide conjugate vaccine. Strains with serogroups C, W and X continue to cause epidemics. Protein antigens in licensed serogroup B vaccines are shared among serogroup B and non-B strains., Purpose: Compare serum bactericidal antibody responses elicited by an investigational native outer membrane vesicle vaccine with over-expressed Factor H binding protein (NOMV-FHbp) and a licensed serogroup B vaccine (MenB-4C) against African serogroup A, B, C, W and X strains., Methods: Human Factor H (FH) transgenic mice were immunized with NOMV-FHbp prepared from a mutant African meningococcal strain containing genetically attenuated endotoxin and a mutant sub-family B FHbp antigen with low FH binding, or with MenB-4C, which contains a recombinant sub-family B FHbp antigen that binds human FH, and three other antigens, NHba, NadA and PorA P1.4, capable of eliciting bactericidal antibody., Results: The NOMV-FHbp elicited serum bactericidal activity against 12 of 13 serogroup A, B, W or X strains from Africa, and four isogenic serogroup B mutants with sub-family B FHbp sequence variants. There was no activity against a serogroup B mutant with sub-family A FHbp, or two serogroup C isolates from a recent outbreak in Northern Nigeria, which were mismatched for both PorA and sub-family of the FHbp vaccine antigen. For MenB-4C, NHba was expressed by all 16 African isolates tested, FHbp sub-family B in 13, and NadA in five. However, MenB-4C elicited titers ≥ 1:10 against only one isolate, and against only two of four serogroup B mutant strains with sub-family B FHbp sequence variants., Conclusions: NOMV-FHbp has greater potential to confer serogroup-independent protection in Africa than the licensed MenB-4C vaccine. However, the NOMV-FHbp vaccine will require inclusion of sub-family A FHbp for coverage against recent serogroup C strains causing outbreaks in Northern Nigeria., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Alcohol-use disorder (AUD) is prevalent and associated with substantial socioeconomic costs. While heritability estimates of AUD are ∼50%, identifying specific gene variants associated with risk for AUD has proven challenging despite considerable investment. Emerging research into heritability of complex diseases has implicated transmission of epigenetic variants in the development of behavioral phenotypes, including drug preference and drug-induced behavior. Several recent rodent studies have specifically focused on paternal transmission of epigenetic variants, which is especially relevant because sires are not present for offspring rearing and changes to offspring phenotype are assumed to result from modifications to the sperm epigenome. While considerable interest in paternal transmission of epigenetic variants has emerged recently, paternal alcohol exposures have been studied for 30+ years with interesting behavioral and physiologic effects noted on offspring. However, only recently, with improvements in technology to identify epigenetic modifications in germ cells, has it been possible to identify mechanisms by which paternal ethanol exposure alters offspring behavior. This review presents an overview of epigenetic inheritance in the context of paternal ethanol exposure and suggests future studies to identify specific effects of paternal ethanol exposure on offspring behavior and response to ethanol., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The effect of protein-based meningococcal vaccines on prevention of nasopharyngeal colonization has been difficult to investigate experimentally because a reliable animal colonization model did not exist., Methods: Human CEACAM1 transgenic mice, which can be colonized by meningococci, were immunized IP with one of two meningococcal native outer membrane vesicle (NOMV) vaccines prepared from mutants with attenuated endotoxin (lpxL1 knockout) and over-expressed sub-family B Factor H-binding proteins (FHbp). Animals were challenged intranasally two weeks after the third dose with wild-type strain H44/76, or were treated IP with anti-NOMV serum before and during the bacterial challenge., Results: The NOMV-1 vaccine, prepared from the serogroup B H44/76 mutant, elicited ∼40-fold higher serum bactericidal antibody titers against the wild-type H44/76 challenge strain than the NOMV-2 vaccine prepared from a heterologous serogroup W mutant strain with different PorA and FHbp amino acid sequence variants. Compared to aluminum hydroxide-immunized control mice, the efficacy for prevention of any H44/76 colonization was 93% (95% confidence interval, 52-99, P<0.0001) for the NOMV-1 vaccine, and 19% (-3-36, P=0.23) for NOMV-2. NOMV-2-vaccinated mice had a 5.6-fold decrease in geometric mean CFU of bacteria per animal in tracheal washes compared to control mice (P=0.007). The efficacy of passive administration of serum from NOMV-1-vaccinated mice to immunologically naïve mice against colonization was 44% (17-61; P=0.002)., Conclusions: Both NOMV vaccines protected against meningococcal colonization but there was greater protection by the NOMV-1 vaccine with antigens matched with the challenge strain. Meningococcal vaccines that target protein antigens have potential to decrease colonization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Curcumin is a plant-derived polyphenol that displays antitumor properties. Incubation of cultured SF-767 glioma cells with curcumin gave rise to intense intranuclear foci of curcumin fluorescence. In vitro studies revealed that nuclear homing by curcumin is not a result of DNA/chromatin binding. On the other hand, curcumin fluorescence colocalized with nucleophosmin, a nucleolus marker protein. To determine the temporal relationship between curcumin-induced apoptosis and nucleolar homing, confocal live cell imaging was performed. The data show that curcumin localization to the nucleolus occurs prior to cell surface exposure of phosphatidylserine. In studies of the mechanism of curcumin-induced apoptosis in SF-767 cells, its effect on the subcellular location of p14(ARF) was determined. Whereas p14(ARF) was confined to the nucleolus in untreated cells, 2 h following incubation with curcumin, it displayed a diffuse nuclear distribution. Given the role of nuclear p14(ARF) in binding the E3 ubiquitin ligase, mouse double minute 2 homolog (MDM2), the effect of curcumin treatment on cellular levels of the tumor suppressor protein, p53, was examined. Between 2 and 4 h following curcumin treatment, p53 levels increased with maximum levels reached by 8 h. Thus, curcumin homing to the nucleolus induces redistribution of p14(ARF) to the nucleoplasm where interaction with MDM2 leads to stabilization of p53, with subsequent initiation of apoptosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Small noncoding RNAs (sncRNAs) mediate a variety of cellular functions in animals and plants. Deep sequencing has made it possible to obtain highly detailed information on the types and abundance of sncRNAs in biological specimens, leading to the discovery that sncRNAs circulate in the blood of humans and mammals. The most abundant types of circulating sncRNAs are microRNAs (miRNAs), 5' transfer RNA (tRNA) halves, and YRNA fragments, with minute amounts of other types that may nevertheless be significant. Of the more abundant circulating sncRNAs only miRNAs have well described functions, but characteristics of the others suggest specific processing and secretion as complexes that protect the RNA from degradation. The properties of circulating sncRNAs are consistent with their serving as signaling molecules, and investigations of circulating miRNAs support the view that they can enter cells and regulate cellular functions. The serum levels of specific sncRNAs change markedly with age, and these changes can be mitigated by calorie restriction (CR), indicating that levels are under physiologic control. The ability of circulating sncRNAs to transmit functions between cells and to regulate a broad spectrum of cellular functions, and the changes in their levels with age, implicate them in the manifestations of aging. Our understanding of the functions of circulating sncRNA, particularly in relation to aging, is currently at a very early stage; results to date suggest that more extensive investigation will yield important insights into mechanisms of aging., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Meningococci bind human fH to down-regulate complement, which enhances survival of the bacteria in serum. A major fH ligand is the vaccine candidate, factor H-binding protein (fHbp). Although fHbp has been considered an essential meningococcal virulence factor, rarely, invasive isolates with absent fHbp genes or frameshift mutations have been identified. In previous studies fH binding to these isolates was not detected. The aim of the present study was to investigate fH binding and complement evasion by invasive meningococcal serogroup B clinical isolates with absent fHbp genes or frameshift mutations. Four of the seven isolates tested bound human fH by flow cytometry and survived in IgG-depleted human serum. In all four, fH binding was decreased after inactivating the gene encoding NspA. Binding of fH to fHbp and NspA is specific for human fH. To investigate fH-dependent evasion of host defenses, human fH transgenic infant rats, or control littermates negative for human fH, were challenged IP with 10(3)-10(4)CFU of two of the isolates with no detectable fH binding by flow cytometry. At 6h, bacteremia caused by both strains was higher in human fH transgenic rats than in control rats (P<0.002). In conclusion, six of the seven isolates had evidence of fH binding and/or human fH-dependent complement evasion in transgenic rats. In four, NspA was as an alternative fH ligand. fHbp vaccination may select for mutants that do not require fHbp for complement evasion. Inclusion of additional target antigens in vaccines containing fHbp may delay emergence of these mutants., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and the control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies., (Published by Elsevier Inc.)

Developing materials that are effective against sexually transmitted pathogens such as Chlamydia trachomatis (Ct) and HIV-1 is challenging both in terms of material selection and improving bio-membrane and cellular permeability at desired mucosal sites. Here, we engineered the prokaryotic bacterial virus (M13 phage) carrying two functional peptides, integrin binding peptide (RGD) and a segment of the polymorphic membrane protein D (PmpD) from Ct, as a phage-based material that can ameliorate Ct infection. Ct is a globally prevalent human pathogen for which there are no effective vaccines or microbicides. We show that engineered phage stably express both RGD motifs and Ct peptides and traffic intracellularly and into the lumen of the inclusion in which the organism resides within the host cell. Engineered phage were able to significantly reduce Ct infection in both HeLa and primary endocervical cells compared with Ct infection alone. Polyclonal antibodies raised against PmpD and co-incubated with constructs prior to infection did not alter the course of infection, indicating that PmpD is responsible for the observed decrease in Ct infection. Our results suggest that phage-based design approaches to vector delivery that overcome mucosal cellular barriers may be effective in preventing Ct and other sexually transmitted pathogens., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A meningococcal group B vaccine containing multiple protein antigens including factor H binding protein (fHbp) and Neisserial heparin binding antigen (NHba) is in clinical development. The ability of antibodies against individual antigens to interact and augment protective immunity is unknown. We assayed human complement-mediated bactericidal activity (SBA) in stored sera from six immunized adults before and after depletion of antibodies to fHbp and/or NHba. All six subjects developed ≥ 4-fold increases in SBA titer against a test strain with fHbp in the variant 1 group with an amino acid sequence that matched the vaccine antigen (GMT <1:4 baseline, to 1:139 after 3 doses of vaccine). By adsorption 88 to >95% of the SBA was directed against fHbp. Four subjects developed ≥ 4-fold increases in SBA titer against a test strain with a heterologous fHbp variant 2 antigen and a homologous NHba amino acid sequence that matched the vaccine antigen (GMT <1:4 baseline, to 1:45). SBA was directed primarily against NHba in one subject, against fHbp in a second, while depletion of either anti-NHba or anti-fHbp antibody removed the majority of SBA in sera from two subjects. In all four subjects, depletion of both anti-fHbp and anti-NHba antibodies removed more SBA than depletion of either antibody individually. Mixing a mouse non-bactericidal anti-fHbp variant 1 antiserum with a mouse anti-NHba antiserum also augmented the anti-NHba SBA titer against this test strain. For meningococcal vaccines that target relatively sparsely exposed antigens such fHbp or NHba, non-bactericidal antibodies against individual antigens can cooperate and elicit SBA., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Meningococcal factor H-binding protein (fHbp) is a promising vaccine candidate that elicits serum bactericidal antibodies in humans. Based on sequence variability of the entire protein, fHbp has been divided into three variant groups or two sub-families. We recently reported that the fHbp architecture was modular, consisting of five variable segments, each encoded by genes from one of two lineages. Based on combinations of segments from different lineages, all 70 known fHbp sequence variants could be classified into one of six modular groups. In this study, we analyzed sequences of 172 new fHbp variants that were available from public databases. All but three variants could be classified into one of the six previously described modular groups. Among systematically collected invasive group B isolates from the U.S. and Europe, modular group I overall was most common (60%) but group IV (natural chimeras) accounted for 23% of UK isolates and <1% of U.S. isolates (P<0.0001). Mouse antisera to recombinant fHbp from each of the modular groups showed modular group-specific bactericidal activity against strains with low fHbp expression but had broader activity against strains with higher fHbp expression. Thus both modular group and relative expression of fHbp affected strain susceptibility to anti-fHbp bactericidal activity. The results confirmed the modular architecture of fHbp and underscored its importance for the design of broadly protective group B vaccines in different regions., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Killing of Neisseria meningitidis can result from complement-mediated serum bactericidal activity (SBA) or opsonophagocytosis (OPA), or a combination of the two mechanisms. While SBA titers > or =1:4 confer protection, recent evidence suggests that this threshold titer may not be required. For example, the incidence of meningococcal disease declines between ages 1 and 4 years without evidence of acquisition of SBA titers > or =1:4. Meningococcal polysaccharide vaccination also elicited OPA and lowered the risk of disease in patients with late complement component deficiencies whose sera did not support SBA. Sera from healthy adults immunized with an outer membrane vesicle vaccine showed OPA killing of N. meningitidis with C6-depleted complement, and whole blood from complement-sufficient non-immunized adults with SBA titers <1:4 also frequently had killing activity. Collectively the data indicate that SBA titers <1:4 and/or vaccine-induced OPA can confer protection against meningococcal disease.

Both Neisseria gonorrhoeae and N. meningitidis bind to factor H which enhances their ability to evade complement-dependent killing. While porin is the ligand for human fH on gonococci, meningococci use a lipoprotein called factor H binding protein (fHbp) to bind to factor H and enhance their ability to evade complement-dependent killing. This protein is currently being intensively investigated as a meningococcal vaccine candidate antigen. Consistent with the observation that meningococci cause natural infection only in humans, the organism resists human complement, and are more readily killed by complement from lower animals. This human species-specific complement evasion has important implications for evaluation of vaccine-elicited antibodies using non-human complement sources and development of animal models of disease.

Protective antigen (PA) is the cell surface recognition moiety of the Bacillus anthracis A-B toxin system, and the active immunogenic component in the currently licensed human anthrax vaccine (BioThrax, or AVA). The serum antibody response to the PA protein is polyclonal and complex both in terms of the antibody combining sites utilized to bind PA and the PA-associated epitopes recognized. We have cloned, sequenced, and expressed a large panel of PA-specific human monoclonal antibodies from seven AVA-immunized donors. Dot blots, Western blots, and radiolabeled antigen capture assays employing both proteolytic fragments of PA and engineered PA sub-domain fusion proteins were used to determine the region (domain) of the PA monomer to which each of the cloned human antibodies bound. The domain specificity of the isolated monoclonals was highly biased towards the amino-terminal 20kDa fragment of PA (PA(20)), with the majority (62%) of independently arising antibody clones reacting with determinants located on this PA fragment. A similar bias in domain specificity was also demonstrated in the serum response of AVA-vaccinated donors. Since PA(20) is cleaved from the remainder of the monomer rapidly following cell surface binding and has no known role in the intoxication process, the immunodominance of PA(20)-associated epitopes may directly affect the efficacy of PA-based anthrax vaccines.

Several studies have demonstrated aberrations in the Electron Transport Complexes (ETC) and Krebs (TCA) cycle in Alzheimer's disease (AD) brain. Optimal activity of these key metabolic pathways depends on several redox active centers and metabolites including heme, coenzyme Q, iron-sulfur, vitamins, minerals, and micronutrients. Disturbed heme metabolism leads to increased aberrations in the ETC (loss of complex IV), dimerization of APP, free radical production, markers of oxidative damage, and ultimately cell death all of which represent key cytopathologies in AD. The mechanism of mitochondrial dysfunction in AD is controversial. The observations that Abeta is found both in the cells and in the mitochondria and that Abeta binds with heme may provide clues to this mechanism. Mitochondrial Abeta may interfere with key metabolites or metabolic pathways in a manner that overwhelms the mitochondrial mechanisms of repair. Identifying the molecular mechanism for how Abeta interferes with mitochondria and that explains the established key cytopathologies in AD may also suggest molecular targets for therapeutic interventions. Below we review recent studies describing the possible role of Abeta in altered energy production through heme metabolism. We further discuss how protecting mitochondria could confer resistance to oxidative and environmental insults. Therapies targeted at protecting mitochondria may improve the clinical outcome of AD patients.

A broadly protective vaccine against meningococcal group B disease is not available. We previously reported that an outer membrane vesicle (OMV) vaccine containing over-expressed genome-derived antigen (GNA) 1870 elicited broader protective antibody responses than recombinant GNA1870 or conventional OMV vaccines prepared from a strain that naturally expresses low amounts of GNA1870. Certain wildtype strains such as H44/76 naturally express larger amounts of GNA1870 and, potentially, could be used to prepare an improved OMV vaccine without genetic over-expression of the antigen. We transformed H44/76 with a shuttle vector to over-express variant 1 (v.1) GNA1870 and compared the immunogenicity in mice of OMV vaccines prepared from wildtype H44/76 (v.1), the mutant, and a recombinant v.1 GNA1870 vaccine. Mice immunized with OMV with over-expressed GNA1870 developed broader serum bactericidal and/or greater C3 deposition activity on the surface of encapsulated strains of N. meningitidis than control mice immunized with the OMV vaccine prepared from the wildtype strain, or the rGNA1870 vaccine. When a panel of group B strains from patients in California was tested, sera from mice immunized with the OMV vaccine containing over-expressed GNA1870 were bactericidal against 100% of the v.1 strains. In contrast, only 20% of isolates that expressed subvariants of the v.1 GNA1870 protein were susceptible to bactericidal activity of antibodies elicited by the rGNA1870 or conventional OMV vaccines. Thus, even a modest increase in GNA1870 expression in a strain that naturally is a high producer of GNA1870 results in an OMV vaccine that elicits broader protection against meningococcal disease.

Mitochondrial oxidative decay, which is a major contributor to aging, is accelerated by many common micronutrient deficiencies. One major mechanism is inhibition of the pathway of heme biosynthesis in mitochondria, which causes a deficit of heme-a. Heme-a, only found in Complex IV, is selectively diminished, resulting in oxidant leakage and accelerated mitochondrial decay, which leads to DNA damage, neural decay, and aging. We emphasize those deficiencies, which appear to cause damage through this mechanism, particularly minerals such as iron (25% of menstruating women ingest <50% of the RDA) or zinc (10% of the population ingest <50% of the RDA). Several vitamin deficiencies, such as biotin or pantothenic acid, also increase mitochondrial oxidants through this mechanism. Additionally, other minerals such as magnesium and manganese that play a role in mitochondrial metabolism, but do not affect heme directly, are discussed. An optimum intake of micronutrients could tune up metabolism and give a marked increase in health, particularly for the poor, elderly, and obese, at little cost.

Background: One dose of a quadrivalent meningococcal conjugate (MC-4) vaccine elicits higher group C bactericidal responses in 2-year olds than a U.S.-licensed quadrivalent polysaccharide (MPS-4) vaccine [Granoff DM, Harris SL. Protective activity of group C anticapsular antibodies elicited in 2-year olds by an investigational quadrivalent Neisseria meningitidis-diptheria toxoid conjugate vaccine. Pediatr Infect Dis J, 2004;23:490-7]., Objective: Assess immunogenicity and persistence of serum antibody to capsular groups A, Y and W-135 in 2-year old children immunized with MC-4 vaccine., Design: Measurement of antibody responses in sera from a multicenter randomized trial comparing MC-4 and MPS-4 vaccines., Results: At 1 month, the group A serum bactericidal GMT was higher in the MC-4 than the MPS-4 group (P < 0.001) but there were no significant differences at 6 months. At 6 months, the respective Y and W-135 GMTs were higher in the MC-4 group (P < 0.05) but there were no differences at 1 month. The higher W-135 bactericidal titers reflected higher antibody avidity in the MC-4 group (P < 0.0001) and avidity maturation between 1 and 6 months (P < 0.05). At 2-year post-vaccination, the proportion of MC-4 immunized children with bactericidal titers > or =1:4 (presumed to be protective) was 15.0% (group A), 32.5% (group Y) and 45% (group W-135), as compared with 2.5, 15.4 and 17.5%, respectively, in unvaccinated children (P < 0.01 for group W-135; P < 0.05 for group A, and P = 0.07 for Y)., Conclusions: One dose of the MC-4 vaccine in 2-year olds elicits higher A, Y and W-135 bactericidal titers than MPS-4 vaccine. Compared with unvaccinated children, serum antibody titers remain elevated for 2 years after MC-4 vaccination. However, many vaccinated children have titers <1:4 and may require a booster dose for sustained protection.

We previously have shown that conjugation of C3d to pneumococcal serotype type 14 capsular polysaccharide (PPS14) significantly enhances anti-PPS14 antibody production to a degree similar to that found when the T-dependent protein carrier ovalbumin (OVA) is coupled to PPS14. However, the anti-PPS14 antibody response to PPS14-C3d conjugates is characterized by less switching from IgM to IgG and lower serum concentrations of anti-PPS14 IgG after secondary immunization. To determine if these quantitative differences in anti-PPS14 IgG are accompanied by qualitative differences in the IgG anti-PPS14 antibodies, we performed several functional assays on serum IgG anti-PPS14 antibodies from mice immunized with PPS14-C3d or PPS14-OVA. Compared with antibodies elicited by immunization with PPS14-C3d, IgG anti-PPS14 antibodies produced after immunization with PPS14-OVA were found to have higher avidity and enhanced function as opsonins. Comparisons of avidity for IgG from serum samples obtained after primary and secondary immunization demonstrated a higher degree of avidity maturation after immunization with PPS14-OVA than with PPS14-C3d. These results suggest that PPS14-C3d conjugates are unlikely to be more efficacious than PPS14 conjugate vaccines incorporating T-dependent protein carriers.

Heme, the major functional form of iron, is synthesized in the mitochondria. Although disturbed heme metabolism causes mitochondrial decay, oxidative stress, and iron accumulation, all of which are hallmarks of ageing, heme has been little studied in nutritional deficiency, in ageing, or age-related disorders such as Alzheimer's disease (AD). Biosynthesis of heme requires Vitamin B(6), riboflavin, biotin, pantothenic acid, and lipoic acid and the minerals zinc, iron, and copper, micronutrients are essential for the production of succinyl-CoA, the precursor for porphyrins, by the TCA (Krebs) cycle. Only a small fraction of the porphyrins synthesized from succinyl-CoA are converted to heme, the rest are excreted out of the body together with the degradation products of heme (e.g. bilirubin). Therefore, the heme biosynthetic pathway causes a net loss of succinyl-CoA from the TCA cycle. The mitochondrial pool of succinyl-CoA may limit heme biosynthesis in deficiencies for micronutrients (e.g. iron or biotin deficiency). Ageing and AD are also associated with hypometabolism, increase in heme oxygenase-1, loss of complex IV, and iron accumulation. Heme is a common denominator for all these changes, suggesting that heme metabolism maybe altered in age-related disorders. Heme can also be a prooxidant: it converts less reactive oxidants to highly reactive free radicals. Free heme has high affinity for different cell structures (protein, membranes, and DNA), triggering site-directed oxidative damage. This review discusses heme metabolism as related to metabolic changes seen in ageing and age-related disorders and highlights the possible role in iron deficiency.

The oxidized DNA base 8-oxoguanine has been commonly measured by enzymatic digestion of DNA to nucleosides followed by high-performance liquid chromatography (HPLC) separation of the adduct 8-oxodeoxyguanosine. There has recently been an enormous debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-oxoguanine can occur at numerous stages in sample preparation. Hence, we have designed an alternative protocol to traditional enzymatic digestion of DNA which (i) limits the potential for artifactual oxidation, (ii) speeds up the assay markedly, (iii) increases the assay's sensitivity moderately, and (iv) addresses criticisms that have been raised concerning the efficiency of DNA digestion by nucleases. In short, we use the Escherichia coli repair enzyme formamidopyrimidine (Fapy) glycosylase to release the base 8-oxoguanine from full-length DNA, then separate 8-oxoguanine from high molecular weight molecules by ultrafiltration (10,000 Da exclusion) and analyze the base adduct by reverse-phase HPLC. Benefits of this approach include (i) rapid removal of the roughly million-fold molar excess of unaltered bases from the sample, (ii) reduction in the length of enzymatic incubations and the number of steps, (iii) elimination of high temperature incubation, (iv) a very clean chromatographic separation, and (v) rapid elution of the analyte and correspondingly greater throughput. Using this improved method, we have followed the induction of 8-oxoguanine in the DNA of peroxide-treated HeLa cells, an experiment that had proved cumbersome with traditional methods.

A family of non-coding sequences in the mRNA (iso-IREs [iron-responsive elements]) regulate synthesis of key proteins in animal iron and oxidative metabolism such as ferritin and mitochondrial aconitase. Differential recognition between iso-IREs and iso-IRPs (iron regulatory proteins) regulates the translation or degradation of the IRE-containing mRNAs. IREs are hairpin loop structures with an internal loop/bulge or bulge that influence the binding of the iso-IRPs. The iso-IRPs have sequence homology to the aconitases and at least one IRP can be converted to an aconitase. Signals that target the iso-IRE/iso-IRP interactions in mRNA include environmental iron, O2, nitric oxide, H2O2, ascorbate, growth factors, and protein kinase C-dependent IRP phosphorylation. Iso-IRE structural specificity suggests a means of pharmacologically targeting mRNA function with chemicals such as Fe-bleomycin and other transition metal complexes that could be extended to other mRNAs with specific structures. With the iso-IRE/iso-IRP system, nature has evolved coordinated combinatorial control of iron and oxygen metabolism that may exemplify control of mRNAs in other metabolic pathways, viral reproduction, and oncogenesis.

To provide a detailed description of the time course and the site specificity of hydroperoxide-induced oxidative stress in red blood cells (RBCs), we have characterized the action of a membrane-soluble (cumene hydroperoxide [cumOOH]) and a water-soluble (hydrogen peroxide [H2O2]) oxidant. The fluorescent polyunsaturated fatty acid (PUFA) parinaric acid (PnA) was used to probe peroxidation processes in the membrane, and oxidation of hemoglobin (Hb) was measured spectrophotometrically as an indicator of cytosolic oxidative stress. The observed degradation patterns of PnA and Hb were clearly distinct for each oxidant. At comparable oxidant concentrations, the cumulative oxidative stress on the RBC membrane was always much higher with cumOOH, whereas much more Hb oxidation was measured with H2O2. The kinetics of Hb oxidation as well as the nature of the products formed were different for each oxidant. The main Hb oxidation product generated gradually by cumOOH was metHb, whereas H2O2 caused the rapid formation of ferrylHb. CumOOH caused more oxidation of endogenous PUFAs and of vitamin E, while the degradation pattern of vitamin E closely resembled that of PnA. At high oxidant concentrations, extensive cell lysis was observed after prolonged incubation. Butylated hydroxytoluene (BHT) completely prevented oxidation of endogenous PUFAs but did not completely prevent hemolysis, indicating that factors other than lipid peroxidation are also important in causing lysis of RBCs. The action of cumOOH is characterized by a gradual reaction with Hb, generating radicals that produce an oxidative stress primarily directed at the membrane, which increases in time to a maximum and then gradually decreases. In contrast, H2O2 crosses the RBC membrane and reacts rapidly with Hb, generating a very reactive radical species that has Hb, not the membrane, as a prime target. H2O2-induced oxidative stress is at a maximum immediately after addition of this oxidant and decreases rapidly to zero in a short time. These findings provide further insight into the mode of action of hydroperoxides and the mechanism of compartmentalization of RBC oxidative damage.

Diagnostic tests for most common hemoglobinopathies and recent advances in structural analysis of variant hemoglobins are reviewed. Routine and newly introduced methods that apply to the diagnosis of sickle cell anemia, thalassemia and the hemoglobin E disorders are presented. A brief description of the clinical course for each of these disorders is given, and potential pitfalls in diagnosis are discussed. Application of high-performance liquid chromatography and various mass spectrometric techniques (electrospray ionization mass spectrometry, liquid secondary ion mass spectrometry, and tandem mass spectrometry) for evaluation of hemoglobinopathy is presented.

Having minimized spectroscopic interference by hemoglobin (Hb), peroxidation processes in intact erythrocytes could be monitored in a continuous assay using the fluorescent polyunsaturated fatty acid, parinaric acid (PnA), as a peroxidation probe. Control experiments to establish the character of the method are described in detail. As a practical application, comparative studies were performed to monitor the response of normal and sickle Hb-containing human erythrocytes to oxidative stress in the PnA assay. After 10 min of incubation with 200 microM cumene hydroperoxide (cumOOH), peroxidation of PnA was found to be enhanced in erythrocytes from sickle cell disease patients (SS: 48 +/- 9% (n = 6) of initial amount had been peroxidized) compared to healthy controls (AA: 30 +/- 4% (n = 9)). PnA peroxidation in erythrocytes from sickle cell trait individuals (AS: 30 +/- 3% (n = 4)) was equal to that in control cells. The increased oxidation of PnA in sickle erythrocytes was accompanied by enhanced oxidation of Hb (metHb and hemichrome formation), indicating that sickle Hb mediates enhanced cumOOH-derived radical generation. It is concluded that PnA can be a useful tool in studying membrane peroxidation processes in intact normal and pathological erythrocytes.

In view of a possible relationship between fish oil, lipid peroxidation, and atherosclerosis, the in vitro lipid peroxidation susceptibility of red blood cells (RBCs) from rabbits on conventional (-FO) and fish oil-enriched diets (+FO) was investigated. The diet caused substantial increases in the RBC concentrations of n-3 polyunsaturated fatty acids (PUFAs), in combination with decreases in the concentration of oleic acid (18:1) and linoleic acid (18:2). Cumene hydroperoxide-induced oxidative stress led to increased overall fatty acid peroxidation in +FO RBCs compared with with -FO RBCs, as quantitated by GLC fatty acid analysis. However, the increased overall susceptibility to lipid peroxidation of +FO RBCs was not reflected in increased peroxidation of every individual fatty acid. This was observed for endogenous arachidonic acid (20:4) as well as, in separate experiments, for exogenously added parinaric acid (PnA). The increased cumene hydroperoxide-induced PUFA oxidation in +FO RBCs was accompanied by a lesser extent of hemolysis. To account for these observations, it is proposed that the increased n-3 PUFA content of +FO RBCs serves as an oxidizable buffer. The present data suggest that oxidation of fatty acids can occur until a critically low level of intact phospholipid in the RBC membrane is reached, after which the membrane destabilizes and hemolysis occurs. At the same time, the PUFA buffer in +FO RBCs could also prevent oxidative damage to specific membrane proteins, which could also help prevent cell lysis.