Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair.

Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT-Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H(2)O(2)) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met-S=O) repair in the entire gray hair follicle. Accordingly, Met-S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT-Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L-methionine. In summary, our data feed the long-voiced, but insufficiently proven, concept of H(2)O(2)-induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.

Transmission photoemission electron microscopy for lateral mapping of the X-ray absorption structure of a metalloprotein in a liquid cell.

We use photoemission electron microscopy in an X-ray transmission mode for full-field imaging of the X-ray absorption structure of copper in the respiratory metalloprotein hemocyanin KLH1. It contains 160 oxygen binding sites. Each site reversibly binds one molecule oxygen between two copper atoms. In our setup, hemocyanin is dissolved in aqueous solution and enclosed in an ultra-high vacuum compatible liquid sample cell with silicon nitride membranes. The local X-ray absorption structure of the liquid sample is converted into photoelectrons at the microscope side of the cell acting as a photocathode. In this way, different copper valencies are laterally distinguished under in vivo-like conditions, attributed to Cu(I) in the deoxy-state and Cu(II) in the oxy-state.

Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 3.

BACKGROUND: Cockroach allergens play a very important role in allergic diseases, especially asthma. The major allergen of the American cockroach (Periplaneta americana), Per a 3, naturally occurs as isoforms of hexamers. OBJECTIVE: The aim of this study was to investigate whether the hexameric structures of Per a 3 influence their allergenicity and immunogenicity. METHODS: Therefore, we compared the different effects of native hexamers and dissociated monomers of cockroach haemolymph (HL), containing almost only Per a 3 proteins (HL-Per a 3), on proliferation and T-helper type 1 (Th1)/Th2 cytokine production of human CD4(+) T cells in co-culture with allergen-pulsed monocyte-derived autologous dendritic cells (DC) as well as the leukotriene release of basophils. RESULTS: In P. americana-sensitized and non-sensitized donors the HL-Per a 3 monomers were internalized faster by immature DC and induced higher proliferation and IFN-gamma production than the hexamers. While in non-sensitized donors IL-4 and IL-5 as well as IL-10 production were also increased after stimulation with monomeric HL-Per a 3-pulsed DC, Th2 cytokine and IL-10 production were only enhanced in P. americana-sensitized donors using hexameric HL-Per a 3-pulsed DC. Furthermore, in the leukotriene release assay the monomers were less effective than the hexamers. CONCLUSION: Our data indicate that the quaternary structure can influence both allergenicity and immunogenicity, also depending on the sensitization status. The monomeric variant of Per a 3 allergens could be a possible candidate for a specific immunotherapy because the IgE-mediated allergic reaction and the Th2-inducing capacity are diminished while the Th1-inducing capacity is retained.

¿Está la cirugía cardíaca garantizada en los niños con síndrome de Down? / No disponible

El 40-50% de los niños con síndrome de Down tienen cardiopatía congénita que pueden ser corregida quirúrgicamente. Existe el riesgo de que haya discriminación contra ellos si existen pocos recursos al considerar que suponen una carga mayor sobre el sistema público de salud o que la cirugía va a tener peores resultados que en los niños sin síndrome de Down. Existe en ciertos medios la creencia de que la contribución que un niño con síndrome de Down hace a la sociedad, percibida como menos importante, no garantiza la utilización de los recursos en este niño, cuando son escasos. En ocasiones se niega la cirugía cardíaca a un niño con SD. El debate persiste si bien los datos demuestran que se va generalizando la oferta de intervención quirúrgica a los niños con síndrome de Down de modo similar a los que no lo tienen. En la actualidad, en África del Sur el Red Cross War Memorial Children´s Hospital (RHX) de patías congénitas. En este Hospital se mantiene que no hay razones importantes para discriminarlos frente a los demás. Pero algunas otras instituciones públicas del país capaces de ofrecer estos servicios quirúrgicos no siguen esta política. Nuestro estudio va a comparar la carga que tienen en relación con la reparación o corrección de la cardiopatía en el RHX. Nuestra revisión va a cuantificar esta carga (en términos de los parámetros que se detallan a continuación) sobre los grupos de niños con y sin síndrome de Down que fueron sometidos a cirugía cardíaca en nuestro hospital durante un período de 5 años (enero 1998- junio 2003). Se recogieron 50 casos de niños con síndrome de Down, cada uno de éstos operado justo después de uno que tenía síndrome de Down. Para valorar la carga generada al sistema público de salud, se analizó el número de días que los niños pasaron en la sala hospitalaria y en la Unidad de Cuidados Intensivos (UCI). El beneficio obtenido por la cirugía cardíaca fue evaluado mediante la determinación del número de días que pasaron en la sala hospitalaria y en la Unidad de cuidados Intensivos (UCI). El beneficio obtenido por la cirugía cardíaca fue evaluado mediante la determinación del número de días que pasaron el en Hospital antes de la primera intervención quirúrgica, entre dos intervenciones (cuando fue necesario realizar dos intervenciones) y después de realizada la última corrección. El cuso postoperatorio fue evaluado mediante la determinación de la frecuencia de reintervenciones, complicaciones de cirugía y tasa de mortalidad precoz en ambos grupos. La mortalidad precoz se definió como muerte tras la cirugía anterior a la salida del hospital. Las admisiones con fines diagnósticos (p.ej., cateterización) fueron incluidas en el número de admisiones, antes o después de la cirugía (AU)

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The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4x6-meric protein of Upogebia pusilla (Crustacea).

The structural properties of the hemocyanin isolated from the Mediterranean mud shrimp, Upogebia pusilla (Decapoda: Thalassinidea), were investigated. Our intent was to make use of the U. pusilla case to perform a structural comparison between crustacean and chelicerate 4x6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4x6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4x6 hemocyanins. Electron micrographs are in agreement with a model in which four 2x6-meric building blocks are arranged in a tetrahedron-like quaternary structure and not in the quasi-square-planar orientation characteristic of the chelicerate protein. Size-exclusion chromatography-fast protein chromatography analysis showed elevated instability of the protein in absence of divalent ions or at pH values higher than 8.0. This analysis also shows that the dissociation of the U. pusilla 4x6-meric hemocyanin into hexamers occurs without any intermediate 2x6-meric state, in contrast with the dissociation profile of the chelicerate protein exhibiting several dissociation intermediates. The oxygen-binding properties of U. pusilla hemocyanin were studied to disclose possible effects by the typical allosteric effectors that modulate the functional properties of crustacean hemocyanin. A marked Bohr and lactate effect, but no significant influence of urate, on the oxygen affinity of U. pusilla hemocyanin were found.

Is cardiac surgery warranted in children with Down syndrome? A case-controlled review.

OBJECTIVES: To compare children with Down syndrome and children without Down syndrome and investigate whether there is a significant difference in the burden that is placed on the health care system between these two groups only in respect of the repair of congenital heart disease at Red Cross War Memorial Children's Hospital, Cape Town, South Africa. DESIGN: This study is a retrospective case control review. SETTING: Red Cross War Memorial Children's Hospital, Cape Town, South Africa. SUBJECTS: The sample group of 50 Down syndrome children who had received cardiac surgery between January 1998 and June 2003 was compared with a control group of 50 nonsyndromic children who had received cardiac surgery during the same period. OUTCOME MEASURES: Sex and diagnoses (cardiac and noncardiac), number of days spent in hospital and in ICU, complication rates, re-operation rates, early mortality rates, planned further cardiac surgery. Costs of these outcomes were not quantified in exact monetary terms. RESULTS: There was no significant difference between the two groups in terms of the burden that was placed on the health care system. Similar complication rates, re-operation rates and early mortality rates were recorded for both groups. The Down syndrome group appeared to benefit more from cardiac surgery than the non-Down syndrome group. CONCLUSION: Denying cardiac surgery to children with Down syndrome does not improve the efficiency of resource allocation. It is therefore not reasonable to suggest that the problem of scarce resources can be ameliorated by discriminating against children with Down syndrome.

Comparison of relative renal function measured with either 99mTc-DTPA or 99mTc-EC dynamic scintigraphies with that measured with 99mTc-DMSA static scintigraphy.

OBJECTIVE: The aim of this study was to compare the renal function measured with either 99mTc-DTPA or 99mTc-EC dynamic scintigraphies with that measured using 99mTc-DMSA static scintigraphy. METHODS: the values of relative renal function measured in 111 renal dynamic scintigraphies performed either with 99mTc-DTPA (55 studies) or with 99mTc-EC (56 studies) were compared with the relative function measured using 99mTc-DMSA static scintigraphy performed within a 1-month period. The comparisons were performed using Wilcoxon signed rank test. The number of 99mTc-DTPA and 99mTc-EC studies that presented relative renal function different by more than 5% from that measured with 99mTc-DMSA, using chi square test were also compared. RESULTS: the relative renal function measured with 99mTc-EC is not statistically different from that measured with 99mTc-DMSA (p = 0.97). The relative renal function measured with 99mTc-DTPA was statistically different from that measured using 99mTc-DMSA, but with a borderline statistical significance (p = 0.05). The number of studies with relative renal function different by more than 5% from that measured with 99mTc-DMSA is higher for the 99mTc-DTPA scintigraphy (p = 0.04) than for 99mTc-EC. CONCLUSION: the relative renal function measured with 99mTc-EC dynamic scintigraphy is comparable with that measured with 99mTc-DMSA static scintigraphy, while the relative renal function measured with 99mTc-DTPA dynamic scintigraphy presents a significant statistical difference from that measured with 99mTc-DMSA static scintigraphy.

Comparison of relative renal function measured with either 99mTc-DTPA or 99mTc-EC dynamic scintigraphies with that measured with 99mTc-DMSA static scintigraphy

OBJECTIVE: The aim of this study was to compare the renal function measured with either 99mTc-DTPA or 99mTc-EC dynamic scintigraphies with that measured using 99mTc-DMSA static scintigraphy. METHODS: the values of relative renal function measured in 111 renal dynamic scintigraphies performed either with 99mTc-DTPA (55 studies) or with 99mTc-EC (56 studies) were compared with the relative function measured using 99mTc-DMSA static scintigraphy performed within a 1-month period. The comparisons were performed using Wilcoxon signed rank test. The number of 99mTc-DTPA and 99mTc-EC studies that presented relative renal function different by more than 5 percent from that measured with 99mTc-DMSA, using chi square test were also compared. RESULTS: the relative renal function measured with 99mTc-EC is not statistically different from that measured with 99mTc-DMSA (p = 0.97). The relative renal function measured with 99mTc-DTPA was statistically different from that measured using 99mTc-DMSA, but with a borderline statistical significance (p = 0.05). The number of studies with relative renal function different by more than 5 percent from that measured with 99mTc-DMSA is higher for the 99mTc-DTPA scintigraphy (p = 0.04) than for 99mTc-EC. CONCLUSION: the relative renal function measured with 99mTc-EC dynamic scintigraphy is comparable with that measured with 99mTc-DMSA static scintigraphy, while the relative renal function measured with 99mTc-DTPA dynamic scintigraphy presents a significant statistical difference from that measured with 99mTc-DMSA static scintigraphy.

Structural properties, conformational stability and oxygen binding properties of Penaeus monodon hemocyanin.

Hemocyanin sequences allineament shows the presence of highly invariant regions especially in the active site and in the tight intersubunits interaction sites. Comparing the aminoacids in contact regions between monomers is possible to interpret the stability of hexamers.

A potential role for water in the modulation of oxygen-binding by tarantula hemocyanin.

Hemocyanin from the tarantula Eurypelma californicum is a large respiratory protein with an exceptional high cooperativity. In contrast to hemocyanins from other species, no physiological allosteric effectors other than protons have been identified so far for this 24-meric oligomer. Here we report for the first time the mediating effects of water activity on the oxygen binding properties of a hemocyanin. Oxygen binding curves were measured in presence of several concentrations of glycine and sucrose since both substances reduce water activity. A pronounced shift of the p(50) was observed in both cases but in different directions: adding sucrose shifts the p(50) towards lower values whereas presence of glycine shows the same tendency as for human hemoglobin. Furthermore, prolonged incubation in sucrose slightly distorts the oxygen binding characteristics of spider hemocyanin. Therefore, only the influence of glycine was further analysed. An analysis based on the nested MWC model indicates, that presence of glycine leads to a preferential population of the two states with lower oxygen affinity (tR and tT) compared to the high affinity states rT and rR. The results corroborate the presence of hierarchically organized interactions in this hemocyanin.

Development of molecular tools for the mulundocandin producer Aspergillus sydowii: DNA-mediated transformation and reporter gene expression.

The echinocandin-type antimycotic mulundocandin and its derivatives are produced by the filamentous fungus Aspergillus sydowii (strain FH2551). These agents have been considered as a potential drug to treat immunocompromised patients who suffer from severe opportunistic fungal infections. In order to generate strains with a modified mulundocandin biosynthesis, we developed molecular tools for genetic engineering of A. sydowii as an alternative to conventional strain improvement procedures. For our experiments, we used strain FH2551, which was discriminated from other Aspergillus strains by determining the sequence of the two internal transcribed spacers (ITS1 and ITS2) of the rDNA locus. In addition, the electrophoretic karyotype of A. sydowii was established using pulsed-field gel electrophoresis (PFGE), leading to a calculated genomic size of about 40 Mb. For gene mapping, chromosomes were subjected to PFGE either unrestricted or after incubation with rare cutting enzymes and probed with heterologous genes. Using the bacterial hygromycin B phosphotransferase gene as a selectable marker for transformation of A. sydowii, we generated transformants with single and multiple copies of plasmid DNA. Subsequently, the heterologous lacZ and gfp genes were efficiently transferred and expressed in A. sydowii. The majority of lacZ-transformants showed more than 6 pkat beta-galactosidase activity/mg protein, while the control strains had no significant background activity. Fluorescence microscopy of gfp-transformants demonstrated that the green-fluorescent protein is present in a stable and active form in the cytoplasm of vegetative hyphae and conidiospores.

Two-photon excitation microscopy of tryptophan-containing proteins.

We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

Vitality status of microorganisms in infected human root dentine.

AIM: This experimental study was initiated to establish a method for characterizing the vitality status of bacteria in infected human root dentine by differentiating between viable and dead microorganisms. METHODOLOGY: Twenty-four root segments of extracted human teeth were infected with either Streptococcus sanguinis or Enterococcus faecalis for 8 weeks. Baseline samples from root dentine (rd) were collected after 4 weeks. These were compared with samples taken at week 8 (control group: n = 12) and with samples collected at week 12 after calcium hydroxide treatment for four weeks (test group: n = 12). After marking viable and dead bacterial cells by two fluorescent dyes, the portion of viable bacteria (PVB) was determined, as well as the number of colony-forming units (CFU). RESULTS: Viable and dead bacteria were identified in all "rd" samples. PVBrd values were lower than PVB values of the bacterial suspension in the root canal lumen. In the control group, PVBrd and CFUrd did not markedly differ at week 4 and at week 8, regardless of the strain used. In the test group, viable but non-culturable sanguinis streptococci (mean PVBrd = 27%; CFUrd = 0) were detected, despite calcium hydroxide treatment. The viability of E. faecalis was not affected by calcium hydroxide. CONCLUSIONS: Fluorescence labelling of bacteria in human root dentine gives valuable additional information about their vitality status compared to the parameter CFU. The method may be suitable for following the fate of bacteria in dentinal tubules, for example in the presence of intracanal dressings.

Small-angle neutron scattering reveals an oxygen-dependent conformational change of the immunogen keyhole limpet hemocyanin type 1 (KLH1).

The respiratory protein of the keyhole limpet, Megathura crenulata, the hemocyanin (KLH), commonly used as an immunogen, binds oxygen cooperatively, which implies the existence of different conformations. For the first time, two different conformations of KLH1 were detected upon oxygenation, a deoxy and an oxy state, using small-angle neutron scattering. Rearrangements in the quaternary structure of KLH1 were predicted from the different radii of gyration and the shifts of the minima and maxima in the scattering curves. Upon oxygenation, KLH1 becomes smaller and more compact. Model reconstruction of KLH1 indicates a hollow cylinder with two rings located close to both ends, which move slightly together upon oxygenation.

Pluraflavins, potent antitumor antibiotics from Saccharothrix sp. DSM 12931.

The new pluramycin-type antibiotics pluraflavin A, C43H54N2O14, pluraflavin B, C43H56N2O15, and pluraflavin E, C36H41NO14 were isolated from cultures of the Saccharothrix species DSM 12931. The structures of the novel compounds were elucidated with the aid of 2D NMR and mass spectrometric investigations. The characteristic structural element of pluraflavins A and B is an additional 4-epi-vancosamine unit at position 13 of the anthraquinone-gamma-pyrone ring system. Pluraflavin E has a carboxyl group in this position. Pluraflavin A has a reactive dimethyl epoxide side chain at position 2 of the anthraquinone-gamma-pyrone aglycon, which may explain the high activity of the antibiotic. The outstanding biological characteristic of pluraflavin A is its powerful, organ-dependent cytostatic action: the IC50 in the colon carcinoma proliferation assay is in the subnanomolar range.

Frequent genomic imbalances suggest commonly altered tumour genes in human hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most frequent-occurring malignant tumours worldwide, but molecular changes of tumour DNA, with the exception of viral integrations and p53 mutations, are poorly understood. In order to search for common macro-imbalances of genomic tumour DNA, 21 HCCs and 3 HCC-cell lines were characterized by comparative genomic hybridization (CGH), subsequent database analyses and in selected cases by fluorescence in situ hybridization (FISH). Chromosomal subregions of 1q, 8q, 17q and 20q showed frequent gains of genomic material, while losses were most prevalent in subregions of 4q, 6q, 13q and 16q. Deleted regions encompass tumour suppressor genes, like RB-1 and the cadherin gene cluster, some of them previously identified as potential target genes in HCC development. Several potential growth- or transformation-promoting genes located in chromosomal subregions showed frequent gains of genomic material. The present study provides a basis for further genomic and expression analyses in HCCs and in addition suggests chromosome 4q to carry a so far unidentified tumour suppressor gene relevant for HCC development.

Two types of urate binding sites on hemocyanin from the crayfish Astacus leptodactylus: an ITC study.

The oxygen binding behaviour of hemocyanins from Crustacea is regulated by small organic compounds such as urate and L-lactate. We investigated the binding characteristics of urate and the related compound caffeine to the 2 x 6-meric hemocyanin of A. leptodactylus under fully oxygenated conditions employing isothermal titration calorimetry (ITC). An analysis of urate and caffeine binding based on a model of n identical binding sites resulted in approximately four binding sites for caffeine and eight for urate. This result suggests that the binding process for these effectors is more complex than this most simple model. Therefore, we introduced a number of alternative models. Displacement experiments helped to select the appropriate model. Based on these experiments, at least two different types of binding sites for urate and caffeine exist on the 2 x 6-meric hemocyanin of A. leptodactylus. The two binding sites differ strongly in their specificity towards the two analogues. It can be hypothesized that two different subunit types (beta and gamma) are responsible for the two types of binding sites.

The 22q11.2 deletion: from diversity to a single gene theory.

The 22q11 deletion syndromes are a group of conditions in which a characteristic spectrum of congenital cardiac defects may be associated with a wide range of noncardiological congenital anomalies. These syndromes are all linked by a deletion in the long arm of chromosome 22. Although it is a large deletion, containing many genes, recent advances have led to the belief that the etiology of the diverse abnormalities of these syndromes may be a single gene deletion. This review outlines the historical development of the various "22q deletion syndromes," including the DiGeorge, velocardiofacial, Takao, Cayler, and CATCH-22 syndromes, briefly describes the relevant cardiac embryogenesis, and then explains how a single gene deletion may encompass the full phenotypic spectrum.

Cloning, sequencing, and heterologous expression of the elmGHIJ genes involved in the biosynthesis of the polyketide antibiotic elloramycin from Streptomyces olivaceus Tü2353.

Elloramycin A (1) belongs to a small family of naphthacenequinones characterized by a unique highly hydroxylated cyclohexenone moiety. A cosmid clone 16F4, harboring genes for the production of 1 from Streptomyces olivaceus Tü2353, has been previously isolated. DNA sequence analysis of a 3.2-kb fragment from 16F4 revealed four open reading frames--the elmGHIJ genes. Heterologous expressions of the elmGHI genes in either Escherichia coli or Streptomyces lividans, followed by biochemical characterizations of the ElmGHI proteins, established ElmG as tetracenomycin B2 oxygenase, ElmH as tetracenomycin F1 monooxygenase, and ElmI as tetracenomycin F2 cyclase. These results provide direct biochemical evidence for the hypothesis that the biosynthesis of 1 in S. olivaceus parallels that of tetracenomycin C (2) in Streptomyces glaucescens and support the notion that the biosynthesis of the highly hydroxylated cyclohexenone moiety in other polyketides most likely follows the same paradigm as the tetracenomycin B2 or A2 oxygenase.