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John M Wood

Publications and source records attributed to John M Wood.

At least 19 recordsLinked to original sources
PubMed2006

Butyrylcholinesterase is present in the human epidermis and is regulated by H2O2: more evidence for oxidative stress in vitiligo.

The human epidermis holds the capacity for autocrine cholinergic signal transduction, but the presence of butyrylcholinesterase (BchE) has not been shown so far. Our results demonstrate that this compartment transcribes a functional BchE. Its activity is even higher compared to acetylcholinesterase (AchE). Moreover, we show that BchE is subject to regulation by H(2)O(2) in a concentration-dependent manner as it was recently described for AchE. Epidermal BchE protein expression and enzyme activities are severely affected by H(2)O(2) in vitiligo as previously demonstrated for AchE. Removal/reduction of H(2)O(2) by a pseudocatalase PC-KUS yields normal/increased protein expression and activities. H(2)O(2)-mediated oxidation of methionine residues in BchE was confirmed by FT-Raman spectroscopy. Computer simulation supported major alteration of the enzyme active site and its tetramerisation domain suggesting deactivation of the enzyme due to H(2)O(2)-mediated oxidation. Based on our results we conclude that H(2)O(2) is a major player in the regulation of the cholinergic signal via both AchE and BchE and this signal is severely affected in the epidermis of patients with active vitiligo.

Acetylcholinesterase↗
PubMed2006

Oxidative stress via hydrogen peroxide affects proopiomelanocortin peptides directly in the epidermis of patients with vitiligo.

The human skin holds the capacity for autocrine processing of the proopiomelanocortin (POMC)-derived peptides. Recent data demonstrated the presence and functionality of ACTH, alpha- and beta-melanocyte-stimulating hormone (MSH), and beta-endorphin in the regulation of skin pigmentation, and a role has been put forward for alpha-MSH as an effective antioxidant. In patients with vitiligo, decreased epidermal POMC processing and low alpha-MSH levels were documented previously. These patients accumulate hydrogen peroxide (H2O2) in the 10(-3) M range in their epidermis. Therefore, we examined the involvement of H2O2 on POMC-derived peptides as possible targets for oxidation by this reactive oxygen species. To address this, we employed immunofluorescence labelling, dot blot analysis, Fourier transform Raman spectroscopy, functionality studies, and computer simulation of the peptide structures. We demonstrate H2O2-mediated oxidation of epidermal ACTH, alpha-MSH, and beta-endorphin in vitiligo owing to oxidation of methionine residues in the sequences of these peptides. Moreover, we show that oxidized beta-endorphin loses its function in the promotion of pigmentation in melanocytes. These changes are reversible upon the reduction of H2O2 levels by a pseudocatalase PC-KUS. Moreover, oxidation of alpha-MSH can be prevented by the formation of a 1:1 complex with the abundant cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin. Thus, using vitiligo, we demonstrate that H2O2 can affect pigmentation via epidermal POMC peptide redox homeostasis.

Adrenocorticotropic Hormone↗
PubMed2006

Specific interaction of the diastereomers 7(R)- and 7(S)-tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo.

Pterin-4a-carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)- and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki=2.3-4.9 microM). Kinetic and binding studies, as well as characterization of the pterin-enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin-PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser-251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser-251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo.

Binding Sites↗
PubMed2006

Functioning methionine sulfoxide reductases A and B are present in human epidermal melanocytes in the cytosol and in the nucleus.

Oxidation of methionine residues by reactive oxygen (ROS) in protein structures leads to the formation of methionine sulfoxide which can consequently lead to a plethora of impaired functionality. The generation of methionine sulfoxide yields ultimately a diastereomeric mixture of the S and R sulfoxides. So far two distinct enzyme families have been identified. MSRA reduces methionine S-sulfoxide, while MSRB reduces the R-diastereomer. It has been shown that these enzymes are involved in regulation of protein function and in elimination of ROS via reversible methionine formation besides protein repair. Importantly, both enzymes require coupling to the NADPH/thioredoxin reductase/thioredoxin electron donor system. In this report, we show for the first time the expression and function of both sulfoxide reductases together with thioredoxin reductase in the cytosol as well as in the nucleus of epidermal melanocytes which are especially sensitive to ROS. Since this cell resides in the basal layer of the epidermis and its numbers and functions are reduced upon ageing and for instance also in depigmentation processes, we believe that this discovery adds an intricate repair mechanism to melanocyte homeostasis and survival.

Cell Fractionation↗
PubMed2006

Twenty-three years of hypersensitivity pneumonitis mortality surveillance in the United States.

BACKGROUND: There are few population-based studies addressing hypersensitivity pneumonitis (HP) in the United States. The National Institute for Occupational Safety and Health (NIOSH) has nationally comprehensive longitudinal mortality data that can contribute to a better understanding of the epidemiology of HP. METHODS: The National Center for Health Statistics multiple cause-of-death data were analyzed for the period 1980-2002. Annual death rate was age-adjusted to the 2000 U.S. standard population. Death rate time-trends were calculated using a linear regression model and geographic distribution of death rates were mapped by state and county. Proportionate mortality ratios (PMRs) by usual industry and occupation adjusted for age, sex, and race, were based on data from 26 states reporting industry and occupation during 1985-1999. RESULTS: Overall age-adjusted death rates increased significantly (P < 0.0001) between 1980 and 2002, from 0.09 to 0.29 per million. Wisconsin had the highest rate at 1.04 per million. Among industries, PMR for HP was significantly high for agricultural production, livestock (PMR, 19.3; 95% CI, 14.0-25.9) and agricultural production, crops (PMR, 4.3; 95% CI, 3.0-6.0). Among occupations, PMR for HP was significantly elevated for farmers, except horticulture (PMR, 8.1; 95% CI, 6.4-10.2). CONCLUSIONS: These findings indicate that agricultural industries are closely associated with HP mortality and preventive strategies are needed to protect workers in these industries.

Adolescent↗
PubMed2006

UVA-irradiated pheomelanin alters the structure of catalase and decreases its activity in human skin.

More than 40 years ago Aronoff showed that catalase structure and activity is seriously affected by photo-oxidation of its own substrate, hydrogen peroxide, owing to cleavage of its porphyrin active site. Here we support the results of Maresca et al. and expand on them by using structural modeling of native catalase and its photo-oxidation product, where both methionine and tryptophan residues are oxidized, yielding a deactivated enzyme.

Catalase↗
PubMed2006

Estrogens can contribute to hydrogen peroxide generation and quinone-mediated DNA damage in peripheral blood lymphocytes from patients with vitiligo.

To date there is ample in vivo and in vitro evidence for increased epidermal and systemic hydrogen peroxide (H(2)O(2)) levels in vitiligo, which can be reduced with a topical application of a pseudocatalase-K.U. Schallreuter (PC-KUS) leading to the recovery of epidermal catalase levels as well as other enzymes in peripheral blood cells. Recently, the generation of H(2)O(2) by oxidative metabolism of estrogens and other aromatic steroids was documented. Therefore, it was tempting to follow estrogen-generated H(2)O(2) and its possible effect on DNA damage in peripheral blood lymphocytes from patients with vitiligo before and after the reduction of epidermal H(2)O(2) with pseudocatalase PC-KUS compared to controls. For this purpose, 20 Caucasian patients were grouped in treated responders (group A, n=11) and untreated active/acute disease (group B, n=9) and compared to Caucasian healthy controls (group C, n=7). Consequently, epidermal catalase protein expression in full skin biopsies was assessed using immunofluorescence labelling together with determination of basal H(2)O(2) levels in peripheral blood lymphocytes. To test the influence of estrogen on H(2)O(2) generation and DNA damage, freshly prepared peripheral blood lymphocytes from all three groups were used for the alkaline comet assay in the presence and absence of catalase. The results of this study demonstrated that reduction of epidermal H(2)O(2) leads to both increased epidermal catalase protein expression as well as decreased H(2)O(2) concentrations in lymphocytes. Moreover, a direct estrogen-mediated DNA damage was identified in both patient groups, which was absent in healthy controls. This effect was not abolished by catalase pointing to direct quinone-mediated DNA damage by estrogens in peripheral blood lymphocytes in vitiligo.

Adult↗
PubMed2006

Computer simulation of native epidermal enzyme structures in the presence and absence of hydrogen peroxide (H2O2): potential and pitfalls.

The human epidermis is especially vulnerable to oxidative stress, which in turn leads to oxidation of important antioxidant enzymes, other proteins, and peptides. Molecular dynamic computer modelling is a new powerful tool to predict or confirm oxidative stress-mediated structural changes consequently altering the function of enzymes/proteins/peptides. Here we used examples of important epidermal antioxidant enzymes before and after hydrogen peroxide (H(2)O(2))-mediated oxidation of susceptible amino-acid residues (i.e. tryptophan, methionine, cysteine, and selenocysteine), which can affect enzyme active sites, cofactor binding, or dimerization/tetramerization domains. Computer modelling predicts that enzyme active sites are altered by H(2)O(2)-mediated oxidation in thioredoxin reductase (TR) and acetylcholinesterase (AchE), whereas cofactor nicotinamide adenine dinucleotide phosphate (reduced form) binding is affected in both catalase and TR but not in glutathione peroxidase. Dimerization is prevented in catalase. These structural changes lead to impaired functionality. Fourier transform-Raman- and Fluorescence spectroscopy together with enzyme kinetics support the results. There are limitations of modelling as demonstrated on the AchE substrate-binding domain, where the computer predicted deactivation, which could not be confirmed by enzyme kinetics. Computer modelling coupled with classical biochemical techniques offers a new powerful tool in cutaneous biology to explore oxidative stress-mediated metabolic changes in the skin.

Computer Simulation↗
PubMed2005

Decreased phenylalanine uptake and turnover in patients with vitiligo.

The human epidermis has the full machinery for autocrine L-phenylalanine turnover to L-tyrosine in keratinocytes and melanocytes. Phenylalanine hydroxylase (PAH) activities increase linearly with inherited skin colour (skin phototype I-VI, Fitzpatrick classification) yielding eightfold more activities in black skin compared to white skin. Moreover, UVB irradiation (1 MED) significantly increases epidermal PAH activities 24 h after exposure. Importantly, L-phenylalanine uptake and turnover in the pigment forming melanocytes is vital for initiation of melanogenesis. In this context it was shown that the uptake of this amino acid is regulated by calcium. The depigmentation disorder vitiligo provides a unique model to follow impaired L-phenylalanine turnover in the skin as well as in serum because affected individuals hold an impaired epidermal 6BH4 de novo synthesis/recycling and regulation including low epidermal PAH activities. After overnight fasting and oral loading with L-phenylalanine (100 mg/kg body weight), 29.6% of 970 patients tested (n=287/970) yielded serum phenylalanine/tyrosine ratios >or=4 and 35.3% (n=342/970) had mild to moderate hyperphenylalaninaemia (HPA), while 9.3% (n=90/970) had both serum L-phenylalanine levels >or=2.0 mg/dl and phe/tyr ratios >or=4.0. Isolated HPA was found in 26% (n=252/970), whereas 20.3% had only increased ratios (n=197/970). None of the patients had phenylketonuria and the family history for this metabolic disease was negative. The IQ followed normal Gaussian distribution. In vitro L-phenylalanine uptake/turnover studies on primary epidermal melanocytes originating from these patients demonstrated a significantly decreased calcium dependent L-phenylalanine uptake and turnover compared to healthy control cells. Based on our observation, we would like to propose that phenylalanine uptake/turnover is under tight control by calcium which in turn could offer an additional novel mechanism in the aetiology of HPA.

Adolescent↗
PubMed2005

Generation of influenza vaccine viruses on Vero cells by reverse genetics: an H5N1 candidate vaccine strain produced under a quality system.

Human influenza vaccine reference strains are prepared as required when an antigenically new strain is recommended by WHO for inclusion in the vaccine. Currently, for influenza A, these strains are produced by a double infection of embryonated hens' eggs using the recommended strain and the laboratory strain PR8 which grows to high titre in eggs, in order to produce a high growth reassortant (HGR). HGRs are provided by WHO reference laboratories to the vaccine manufacturing industry which use them to prepare seed virus for vaccine production. The use of reverse genetics in preparing vaccine reference strains offers several advantages over the traditional method: (i) the reverse genetics approach is a direct rational approach compared with the potentially hit-or-miss traditional approach; (ii) reverse genetics will decontaminate a wild type virus that may have been derived in a non-validated system, e.g. a cell line not validated for vaccine purposes, or that may contain additional pathogens; (iii) at the plasmid stage, the HA can be engineered to remove pathogenic traits. The use of reverse genetics in deriving HGRs has been demonstrated by several laboratories, including its use in deriving a non-pathogenic reassortant strain from a highly pathogenic virus. In this report, we have advanced the use of reverse genetics by making use of a cell line acceptable for human vaccine production, by demonstrating directly the short time frame in which a reassortant virus can be derived, and by deriving a non-pathogenic pandemic vaccine reference virus in cells validated for vaccine production and under quality controlled conditions.

Animals↗
PubMed2005

Cross-reactivity to highly pathogenic avian influenza H5N1 viruses after vaccination with nonadjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a potential priming strategy.

Antigenically well-matched vaccines against highly pathogenic avian influenza H5N1 viruses are urgently required. Human serum samples after immunization with MF59 or nonadjuvanted A/duck/Singapore/97 (H5N3) vaccine were tested for antibody to 1997-2004 human H5N1 viruses. Antibody responses to 3 doses of nonadjuvanted vaccine were poor and were higher after MF59-adjuvanted vaccine, with seroconversion rates to A/HongKong/156/97, A/HongKong/213/03, A/Thailand/16/04, and A/Vietnam/1203/04 of 100% (P < .0001), 100% (P < .0001), 71% (P = .0004), and 43% (P = .0128) in 14 subjects, respectively, compared with 27%, 27%, 0%, and 0% in 11 who received nonadjuvanted vaccine. These findings have implications for the rational design of pandemic vaccines against influenza H5.

Adjuvants, Immunologic↗
PubMed2004

Regulation of tyrosinase by tetrahydropteridines and H2O2.

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.

Binding Sites↗
PubMed2004

Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes.

Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24h after UVB light with only one minimal erythemal dose. Since UVB generates also H(2)O(2), we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1x10(-3)M), the V(max) for enzyme activity increased 4-fold by H(2)O(2) (>2.0x10(-3)M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp(120) residue to 5-OH-Trp(120) by H(2)O(2) causing a conformational change in the regulatory domain. PAH was still activated by H(2)O(2) in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H(2)O(2) by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis.

Cell Division↗
PubMed2004

Activation/deactivation of acetylcholinesterase by H2O2: more evidence for oxidative stress in vitiligo.

Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.

Acetylcholinesterase↗
PubMed2004

Confronting the avian influenza threat: vaccine development for a potential pandemic.

Sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. Vaccination is the principal means to combat the impact of influenza. During an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. An emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. The manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. Strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirus-expressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. In clinical trials, conventional surface-antigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. Adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen.

Animals↗
PubMed2004

From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza.

Over the past eight years, cases of human infection with highly pathogenic avian influenza viruses have raised international concern that we could be on the brink of a global influenza pandemic. Many of these human infections have proved fatal and if the viruses had been able to transmit efficiently from person to person, the effects would have been devastating. How can we arm ourselves against this pandemic threat when these viruses are too dangerous to use in conventional vaccine production? Recent technological developments (reverse genetics) have allowed us to manipulate the influenza virus genome so that we can construct safe, high-yielding vaccine strains. However, the transition of reverse-genetic technologies from the research laboratory to the manufacturing environment has presented new challenges for vaccine manufacturers as well as veterinary and public health authorities.

Disease Outbreaks↗
PubMed2003

Safety and antigenicity of whole virus and subunit influenza A/Hong Kong/1073/99 (H9N2) vaccine in healthy adults: phase I randomised trial.

BACKGROUND: In 1999, avian influenza A/Hong Kong/1073/99 (H9N2) virus emerged as a pandemic threat to human beings. We aimed to assess safety, tolerability, and antigenicity of whole virus and subunit H9N2 vaccines in healthy volunteers. METHODS: In a phase I randomised trial we randomly assigned 60 participants to whole virus or subunit H9N2 vaccine. Two doses of 7.5 microg, 15 microg, or 30 microg haemagglutinin influenza A H9N2 vaccine, were given 3 weeks apart. We measured antibody responses by haemagglutination-inhibition and microneutralisation. The primary outcome was geometric mean antibody titre 21 days after vaccination. Analysis was per protocol. FINDINGS: Both vaccines were safe and well tolerated. The antibody titres after vaccination did not differ significantly between subunit and whole virus vaccine. 24 of 60 prevaccination serum samples had unexpected reactivity to H9N2, but only in participants older than 32 years, in whom one dose of either vaccine evoked antibody responses associated with protection. In participants aged 32 years or younger, antibody responses to one dose of whole virus or subunit vaccine were poor, fulfilling none of the criteria used for yearly relicensing of interpandemic vaccines. Whole virus vaccine produced a significantly higher probability of seroconversion compared with subunit virus for this age-group. INTERPRETATION: In immunologically naive patients whole-virus vaccine produced better responses than subunit vaccine. Two doses of subunit or whole virus vaccine would leave a large proportion of the naive population (< or =32 years) unprotected against A/Hong Kong/1073/99 (H9N2). Primed patients should be protected with a single dose of either vaccine.

Adolescent↗