The tactile receptive fields of freely moving Caenorhabditis elegans nematodes.

Sensory neurons embedded in skin are responsible for the sense of touch. In humans and other mammals, touch sensation depends on thousands of diverse somatosensory neurons. By contrast, Caenorhabditis elegans nematodes have six gentle touch receptor neurons linked to simple behaviors. The classical touch assay uses an eyebrow hair to stimulate freely moving C. elegans, evoking evasive behavioral responses. This assay has led to the discovery of genes required for touch sensation, but does not provide control over stimulus strength or position. Here, we present an integrated system for performing automated, quantitative touch assays that circumvents these limitations and incorporates automated measurements of behavioral responses. The Highly Automated Worm Kicker (HAWK) unites a microfabricated silicon force sensor holding a glass bead forming the contact surface and video analysis with real-time force and position control. Using this system, we stimulated animals along the anterior-posterior axis and compared responses in wild-type and spc-1(dn) transgenic animals, which have a touch defect due to expression of a dominant-negative α-spectrin protein fragment. As expected from prior studies, delivering large stimuli anterior and posterior to the mid-point of the body evoked a reversal and a speed-up, respectively. The probability of evoking a response of either kind depended on stimulus strength and location; once initiated, the magnitude and quality of both reversal and speed-up behavioral responses were uncorrelated with stimulus location, strength, or the absence or presence of the spc-1(dn) transgene. Wild-type animals failed to respond when the stimulus was applied near the mid-point. These results show that stimulus strength and location govern the activation of a characteristic motor program and that the C. elegans body surface consists of two receptive fields separated by a gap.

A new depside from Usnea aciculifera growing in Vietnam.

From Usnea aciculifera, a new depside aciculiferin A (1) was isolated, together with eleven known compounds, (+)-(12R)-usnic acid (2), methyl haematommate (3), methyl beta-orsellinate (4), methyl orsellinate (5), atranol (6), 7-hydroxy-5-methoxy-6-methylphthalide (7), norstictic acid (8), stictic acid (9), atranorin (10), barbatinic acid (11) and diffractaic acid (12). Their chemical structures were elucidated by 1D and 2D NMR spectroscopic as well as HR-ESI-MS analysis. Usnic acid (2) and depside diffractaic acid (12) presented in high yield of around 1.5% of the dried material. Some lichen substances inhibited the growth of some cancer cell lines. Three depsides, 1, 11 and 12, were evaluated for their cytotoxic activity against HeLa (human epithelial carcinoma), NCI-H460 (human lung cancer) and MCF-7 (human breast cancer) cell lines at the concentration of 100 microg/mL. Depside 1 showed good and depside 12 strong cytotoxic activity against three surveyed cancer cell lines.

[Appraisal of the optical diagnostic technique for cervical intra-epithelial neoplasia by polarimetric imaging "Polcolpo". Comparison with colposcopic and histological diagnoses by biopsy]. / Étude prospective comparant les performances de la colposcopie classique à la colposcopie guidée par polarimétrie sur la détection des néoplasies intra-épithéliales du col utérin.

OBJECTIVES: The main objective of this study was to compare the performances of polarimetric imaging and standard colposcopy for the detection of CIN. MATERIALS AND METHODS: We performed a monocentric prospective clinical study. The standard colposcopic diagnosis obtained during a first consultation was compared to the diagnosis provided by polarimetric imaging in a second consultation. In addition to the biopsies guided by classical or polarimetric colposcopy, a systematic biopsy taken at a predefined location allowed to calculate the specificities and sensitivities of both techniques. RESULTS: One hundred and forty-one patients were included, all of them with anomalous Pap smears. Sixty-seven cone biopsies were taken, 69 % of which were eventually diagnosed with CIN2+ lesions. The sensitivities and specificities were found to be equal for standard and polarimetric colposcopies. CONCLUSION: We could not demonstrate any improvement of the diagnostic performances with polarimetric colposcopy alone. However, for both healthy and pathological cervices, we observed interesting polarimetric responses involving other characteristics than those we initially assumed, and which will be taken into account in a future study.

[Prevalence of HPV-induced lesions in men: a study from 246 heterosexual couples]. / Prévalence de lésions HPV-induites chez les hommes : étude à partir de 246 couples hétérosexuels.

OBJECTIVES: Human Papillomaviruses (HPV) infection is high in heterosexual couples. We have evaluated clinically the frequency and the histological type of genital lesions in men whose partners have an HPV cervical and/or external genital lesion. PATIENTS AND METHODS: We conducted a cross-sectional study; we examined 246 men whose partner was referred for HPV lesions treatment of either the external genital tract or the cervix. All clinical HPV lesions detected in the men then underwent histological examination. RESULTS: In 72% of cases, the couples were 18 to 35 years old. We detected HPV clinical lesions, confirmed histologically in 43% (106/246) of men. Warts and high-grade intraepithelial neoplasia were diagnosed in 83 (78%) and 23 (22%) of cases, respectively. The prevalence of clinical HPV lesions in men ranged from 34% in case of HG CIN to 80% when the female partner suffered from genital warts. DISCUSSION AND CONCLUSIONS: The high frequency of clinical HPV lesions in men whose the partner has warts should lead to a peniscopy of the partner in these cases. Similarly, the peniscopy detects an HPV lesion in a third of men of which the partner gets a HG CIN. It is necessary to realize prospective studies to reevaluate the impact of diagnosing and treating male lesions with regard to the evolution of HG CIN in their partner.

Molecular epidemiology of Escherichia coli bacteremia in liver transplant recipients.

The characteristics of Escherichia coli strains causing bacteremia in profoundly immunosuppressed patients such as transplant recipients are undefined. The phylogenetic group and the virulence genotype of 57 distinct E. coli strains that caused bacteremia in 53 liver transplant recipients were investigated, and the association of these characteristics with host factors and in-hospital mortality was examined. Phylogenetic groups A, B1, B2, and D accounted for 39%, 10%, 25%, and 26% of the isolates, respectively. The most prevalent virulence genes were fyuA (yersiniabactin system: 70%) and iutA (aerobactin system: 63%), whereas hlyA (alpha-hemolysin) and cnf1 (cytotoxic necrotizing factor 1) occurred in only 14% and 12% of isolates, respectively. Most virulence genes were significantly more prevalent among group B2 and D isolates, vs. group A and B1 isolates. The overall rate of in-hospital mortality after E. coli bacteremia was 20%. Predictors of mortality included onset of bacteremia within 30 days of transplantation or during the intensive care unit stay, and non-urinary source and cutaneous source, but not E. coli phylogenetic group or virulence profile. Compared with historical E. coli bloodstream isolates from non-transplant patients, those from liver transplant recipients are characterized by a higher prevalence of groups A and B1 isolates and reduced virulence gene content. This finding can be explained by the severely immunocompromised status of the patients and the predominance of abdominal-source bacteremic episodes. Time of onset and source of bacteremia, not bacterial characteristics, predict mortality.

Radiographic severity of rheumatoid arthritis in African Americans: results from a multicenter observational study.

OBJECTIVE: To describe radiographic changes in African Americans with rheumatoid arthritis (RA) from the Consortium for the Longitudinal Evaluations of African Americans with Early Rheumatoid Arthritis (CLEAR) Registry, a multicenter observational study. METHODS: Self-declared African American patients were enrolled in CLEAR I, a longitudinal cohort of early RA (disease duration of <2 years) from 2000 to 2005, or in CLEAR II, a cross-sectional cohort (any disease duration) from 2006 to the present. Demographic and clinical data were obtained, and sets of hand/wrist and foot radiographs were scored using the modified Sharp/van der Heijde scoring system. RESULTS: A total of 357 and 418 patients were enrolled in CLEAR I and CLEAR II, respectively. We report here an interim analysis of radiographic severity in these patients. For the CLEAR I cohort, 294 patients had a mean radiographic score of 2.89 at the baseline visit; 32.0% showed either erosions (25.9%) or joint space narrowing (JSN; 19.4%). At the 36-month visit, the mean score was 5.65; 44.2% had erosions, 41.5% had JSN, and 54.4% had either. Among those patients without radiographic damage at baseline, 18.9% had progressed at the 36-month visit, compared with 57.1% of those with baseline damage (P < 0.0001). For the CLEAR II cohort, of 167 patients with RA of any duration, 65.3% exhibited joint erosions, 65.3% exhibited JSN, and 74.8% exhibited either. The mean radiographic score was 33.42. CONCLUSION: To our knowledge, this is the largest radiographic study of African American RA patients. Damage occurs early in the disease and is associated with radiographic progression at 3 years of disease duration. The CLEAR Registry will provide a valuable resource for future analyses of genetic, clinical, and environmental factors associated with radiographic severity of RA in African Americans.

IscA, an alternate scaffold for Fe-S cluster biosynthesis.

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and Mössbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.

Accuracy of thin-layer cytology in patients undergoing cervical cone biopsy.

OBJECTIVE: To compare the accuracy of thin-layer cytology with Autocyte PREP (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.) with conventional smears in 500 women undergoing cervical cone biopsy. STUDY DESIGN: The study was performed among 500 consecutive women presenting for cone biopsy for high grade cervical intraepithelial neoplasia (CIN) on biopsy in 350 (70%) and discrepant cytology/colpohistology in 150 (30%). Before performing a cone biopsy, two cervical samples were collected for conventional smears and thin-layer cytologic slides, with randomization of the order. Conventional smears were stained and diagnosed at Pasteur Cerba, while thin-layer cytologic slides were processed at a local TriPath office (Meylan, France) and sent in a masked fashion for screening at Pasteur Cerba. Any slides initially read as normal were reviewed again and reported without knowledge of the other cytologic or cone biopsy data. The final cytologic diagnoses for the two methods were compared with histopathology of the cone biopsy. RESULTS: The conventional smear was unsatisfactory in 58 (11.6%) of cases, while there were 4 (0.8%) unsatisfactory thin-layer cytologic slides (P < .001). Endocervical cells were missing from 31 (6.2%) of conventional smears and 34 (6.8%) of thin-layer cytologic slides. For the pooled data, sensitivities of conventional smear and thin layer for detecting high grade CIN (0.82% and 0.86%, respectively) were similar as were specificities (0.40% and 0.43%, respectively). When first samples were compared, the sensitivities of the conventional smear and thin layer for high grade CIN were 0.79% and 0.89%, respectively (P = .02), with corresponding specificities of 0.41% and 0.36% (P < .01). CONCLUSION: When controlled for sample order, the sensitivity of thin-layer cytology for detecting high grade CIN was significantly higher than that of conventional smears in patients with previous abnormal cytology, but at the expense of specificity.

Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase.

The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mössbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.

Mössbauer characterization of the iron-sulfur clusters in Desulfovibrio vulgaris hydrogenase.

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S](H)) and a binuclear Fe cluster ([2Fe](H)), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O(2), indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field Mössbauer spectroscopy has been used to characterize the Fe-S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the CO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters, and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S]-[2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S](H) cluster from the 2+ to the 1+ state, followed by transfer of the reducing equivalent from the [4Fe-4S](H) subcluster to the binuclear [2Fe](H) subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S](H) and the [2Fe](H) subclusters. Implication of such a CO binding effect is discussed.

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU.

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and Mössbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.

Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mössbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

Importance of the depressor septi nasi muscle in rhinoplasty: anatomic study and clinical application.

An active depressor septi muscle can accentuate a drooping nasal tip and shorten the upper lip on animation. We have found that dissection and transposition of the depressor septi muscle during rhinoplasty can improve the tip-upper lip relationship in appropriately selected patients. Although the anatomy of the depressor septi muscle has been described, the anatomic variations of this muscle have not been previously reported. The goals of this study were two-fold: (1) to define the anatomic variations of the depressor septi muscle using 55 fresh cadaver dissections and (2) to develop a clinically applicable algorithm for modification of this muscle during rhinoplasty in those patients with a short upper lip and/or tip-upper lip imbalance. Fifty-five fresh cadavers were dissected, and the anatomic variations of the depressor septi muscle were recorded. Three variations of the depressor septi muscle were delineated: type I inserted fully into the orbicularis oris (62 percent); type II inserted into the periosteum and incompletely into the orbicularis oris (22 percent); and type III showed no, or rudimentary, depressor septi muscle (16 percent). Sixty-two patients over a 4-year period (from 1995 to 1999) were identified preoperatively with a hyperactive depressor septi diagnosed by a descending nasal tip and shortened upper lip on animation. These patients underwent dissection and transposition (not resection) of the paired depressor septi during rhinoplasty with improvement or correction of the tip-upper lip imbalance in 88 percent of cases. The anatomic study, surgical indications, rationale for the operative technique, and clinical cases are presented. Dissection and transposition of the depressor septi is a valuable adjunct to rhinoplasty in patients with a type I or II muscle variant.

A short Fe-Fe distance in peroxodiferric ferritin: control of Fe substrate versus cofactor decay?

The reaction of oxygen with protein diiron sites is important in bioorganic syntheses and biomineralization. An unusually short Fe-Fe distance of 2.53 angstroms was found in the diiron (mu-1,2 peroxodiferric) intermediate that forms in the early steps of ferritin biomineralization. This distance suggests the presence of a unique triply bridged structure. The Fe-Fe distances in the mu-1, 2 peroxodiferric complexes that were characterized previously are much longer (3.1 to 4.0 angstroms). The 2.53 angstrom Fe-Fe distance requires a small Fe-O-O angle (approximately 106 degrees to 107 degrees). This geometry should favor decay of the peroxodiferric complex by the release of H2O2 and mu-oxo or mu-hydroxo diferric biomineral precursors rather than by oxidation of the organic substrate. Geometrical differences may thus explain how diiron sites can function either as a substrate (in ferritin biomineralization) or as a cofactor (in O2 activation).

Biochemical and spectroscopic characterization of overexpressed fuscoredoxin from Escherichia coli.

Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfido-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mössbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data.

The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins.

Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench Mössbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S. A., Small, W., Krebs, C., Tavares, P., Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]. To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mössbauer conclusions. The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe. Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.

Pteridine analysis in urine by capillary electrophoresis using laser-induced fluorescence detection.

Pteridines are a class of compounds excreted in urine, the levels of which are found to elevate significantly in tumor-related diseases. For the first time, we have developed a method, based on high-performance capillary electrophoresis (HPCE) and laser-induced fluorescence (LIF) detection, to monitor the pteridine levels in urine. HPCE provides better separation than high-performance liquid chromatography and the LIF detector enables us to detect minute amounts of pteridines in body fluid. Eight different pteridine derivatives were well separated in 0.1 M Tris-0.1 M borate-2 mM EDTA buffer (pH 8.75) using a 60-cm fused-silica capillary (50-micron i.d., 35-cm effective length), six of which were detected and characterized in urine samples from normal persons and different cancer patients. The detection limits of these pteridines are under 1 x 10(-10) M. The levels of neopterin, pterine, xanthopterin, and pterin-6-carboxylic acid were found to be significantly elevated in urine excreted by cancer patents, while the level of isoxanthopterin dropped in these patients. No significant change of biopterin level was found between healthy individuals and cancer patients. This method can be used in clinical laboratories either for cancer monitoring or for precancer screening.

Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen.

The heme domain (iNOS(heme)) of inducible nitric oxide synthase (NOS) was expressed in Escherichia coli and purified to homogeneity. Rapid freeze-quench (RFQ) EPR was used to monitor the reaction of the reduced iNOS(heme) with oxygen in the presence and absence of substrate. In these reactions, heme oxidation occurs at a rate of approximately 15 s(-)(1) at 4 degrees C. A transient species with a g = 2.0 EPR signal is also observed under these conditions. The spectral properties of the g = 2.0 signal are those of an anisotropic organic radical with S = (1)/(2). Comparison of the EPR spectra obtained when iNOS(heme) is reconstituted with N5-(14)N- and (15)N-substituted tetrahydrobiopterin (H(4)B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H(3)B.) of the bound H(4)B. Substitution of D(2)O for H(2)O reveals the presence of hyperfine-coupled exchangeable protons in the H(4)B radical. This radical forms at a rate of 15-20 s(-)(1), with a slower decay rate that varies (0.12-0.7 s(-)(1)) depending on the substrate. At 127 ms, H(3)B. accumulates to a maximum of 80% of the total iNOS(heme) concentration in the presence of arginine but only to approximately 2.8% in the presence of NHA. Double-mixing RFQ experiments, where NHA is added after the formation of H(3)B., show that NHA does not react rapidly with H(3)B. and suggest that NHA instead prevents the formation of the H(4)B radical. These data constitute the first direct evidence for an NOS-bound H(3)B. and are most consistent with a role for H(4)B in electron transfer in the NOS reaction.