Particulate air pollutants, APOE alleles and their contributions to cognitive impairment in older women and to amyloidogenesis in experimental models.

Exposure to particulate matter (PM) in the ambient air and its interactions with APOE alleles may contribute to the acceleration of brain aging and the pathogenesis of Alzheimer's disease (AD). Neurodegenerative effects of particulate air pollutants were examined in a US-wide cohort of older women from the Women's Health Initiative Memory Study (WHIMS) and in experimental mouse models. Residing in places with fine PM exceeding EPA standards increased the risks for global cognitive decline and all-cause dementia respectively by 81 and 92%, with stronger adverse effects in APOE ɛ4/4 carriers. Female EFAD transgenic mice (5xFAD+/-/human APOE ɛ3 or ɛ4+/+) with 225 h exposure to urban nanosized PM (nPM) over 15 weeks showed increased cerebral ß-amyloid by thioflavin S for fibrillary amyloid and by immunocytochemistry for Aß deposits, both exacerbated by APOE ɛ4. Moreover, nPM exposure increased Aß oligomers, caused selective atrophy of hippocampal CA1 neurites, and decreased the glutamate GluR1 subunit. Wildtype C57BL/6 female mice also showed nPM-induced CA1 atrophy and GluR1 decrease. In vitro nPM exposure of neuroblastoma cells (N2a-APP/swe) increased the pro-amyloidogenic processing of the amyloid precursor protein (APP). We suggest that airborne PM exposure promotes pathological brain aging in older women, with potentially a greater impact in ɛ4 carriers. The underlying mechanisms may involve increased cerebral Aß production and selective changes in hippocampal CA1 neurons and glutamate receptor subunits.

Particulate Air Pollutants, Brain Structure, and Neurocognitive Disorders in Older Women.

Introduction: An increasing number of studies have suggested that exposure to particulate matter (PM) may represent a novel - and potentially amendable - environmental determinant of brain aging. The current longitudinal environmental epidemiological study addressed some important knowledge gaps in this emerging field, which combines the study of air pollution and neuroepidemiology. The investigators hypothesized that long-term PM exposure adversely influences global brain volume and brain regions (e.g., frontal lobe or hippocampus) that are critical to memory and complex cognitive processing or that are affected by neuropathological changes in dementia. It was also hypothesized that long-term PM exposure results in neurovascular damage and may increase the risk of mild cognitive impairment (MCI) and -dementia. Methods: The investigators selected a well-characterized and geographically diverse population of older women (N = 7,479; average age = 71.0 ± 3.8 years at baseline) in the Women's Health Initiative (WHI) Memory Study (WHIMS) cohort (1996-2007), which included a subcohort (n = 1,403) enrolled in the WHIMS-Magnetic Resonance Imaging (WHIMS-MRI) study (2005-2006). Residence-specific yearly exposures to PM ≤ 2.5 µm in aerodynamic diameter (PM2.5) were estimated using a Bayesian maximum entropy spatiotemporal model of annual monitoring data (1999-2007) recorded in the U.S. Environmental Protection Agency (U.S. EPA) Air Quality System (AQS). Annual exposures (1996-2005) to diesel PM (DPM) were assigned to each residential census tract in a nationwide spatiotemporal mapping, based on a generalized additive model (GAM), to conduct census tract-specific temporal interpolation of DPM on-road estimates given by the U.S. EPA National-Scale Air Toxics Assessment Program. Multiple linear regression and multicovariate-adjusted Cox models were used to examine the associations, with statistical adjustment for multiple potential confounders. Results: The investigators found that participants had smaller brain volumes, especially in the normal-appearing white matter (WM), if they lived in locations with higher levels of cumulative exposure (1999-2006) to PM 2.5 before the brain MRI scans were performed. The associations were not explained by sociodemographic factors, socioeconomic status, lifestyle factors, or other clinical characteristics. Analyses showed that the adverse effect on brain structure in the participants was driven primarily by the smaller WM volumes associated with cumulative PM2.5 exposures, which were present in the WM divisions of the association brain area (frontal, parietal, and temporal lobes) and corpus callosum. Increased DPM exposures were associated with larger ventricular volume, suggesting an overall atrophic effect on the aging brains. The participants tended to have smaller gray matter (GM) volumes if they lived in areas with the highest (i.e., fourth quartile) estimated cumulative DPM exposure in the 10 years before the brain MRI scans, compared with women in the first to third quartiles. This observed association was present in the total brain GM and in the association brain cortices. The associations with normal-appearing WM varied by DPM exposure range. For women with estimated cumulative exposure below that of the fourth quartile, increased DPM estimates were associated with smaller WM volumes. However, for women with increased cumulative DPM exposures estimates in the fourth quartile, WM volumes were larger. This pattern of association was found consistently in the association brain area; no measurable difference was found in the volume of the corpus callosum. These observed adverse effects of cumulative exposure to PM2.5 (linking exposure with smaller WM volumes) and to DPM (linking exposure in the highest quartile with smaller GM volumes) were not significantly modified by existing cardiovascular diseases, diabetes mellitus, obesity, or measured white blood cell (WBC) count. MRI measurements of the structural brain showed no differences in small-vessel ischemic diseases (SVID) in participants with varying levels of cumulative exposure to PM2.5 (1999-2006) or DPM (1996-2005), and no associations between PM exposures and SVID volumes were noted for total brain, association brain area, GM, or WM. For neurocognitive outcomes followed until 2007, the investigators found no evidence for increased risk of MCI/dementia associated with long-term PM exposures. Although exploratory secondary analyses showed different patterns of associations linking PM exposures separately with MCI and dementia, none of the -results was statistically significant. A similar lack of associations between PM exposures and MCI/dementia was found across the subgroups, with no strong indications for effect modification by cardiovascular diseases, diabetes mellitus, obesity, or WBC count. Conclusions: The investigators concluded that their study findings support the hypothesized brain-structure neurotoxicity associated with PM exposures, a result that is in line with emerging neurotoxicological data. However, the investigators found no evidence of increased risk of MCI/dementia associated with long-term PM exposures.To better test the neurovascular effect hypothesis in PM-associated neurotoxic effects on the aging brain, the investigators recommend that future studies pay greater attention to selecting optimal populations with repeated measurements of cerebrovascular damage and address the possibility of selection biases accordingly. To further investigate the long-term consequence of brain-structure neurotoxicity on pathological brain aging, future researchers should take the pathobiologically heterogeneous neurocognitive outcomes into account and design adequately powered prospective cohort studies with improved exposure estimation and valid outcome ascertainment to assess whether PM-associated neurotoxicity increases the risks of pathological brain aging, including MCI and dementia.

[Multiple epidermolytic acanthomas of the genitalia]. / Hyperkératose épidermolytique génitale (acanthomes épidermolytiques génitaux multiples).

BACKGROUND: Epidermolytic hyperkeratosis presents a particular histological image common to several clinical pictures, including that of keratinopathic ichthyoses. It may also occur fortuitously in various tumoral and inflammatory lesions. It is the elementary histopathological lesion of epidermolytic acanthoma, which may either be single or multiple, and when it occurs in the genital area, is known as epidermolytic hyperkeratosis of the genitalia or multiple epidermolytic acanthoma of the genitalia. Herein, we report two characteristic cases of epidermolytic hyperkeratosis of the genitalia. PATIENTS AND METHODS: The first patient was a 50-year-old woman consulting for vulvar pain in whom clinical examination revealed the presence of multiple papules on the labia majora and minora. The second patient was a 44-year-old man consulting for verrucous lesions of the scrotum. In both cases, biopsy revealed an histopathological aspect identical with acanthosis, hyperkeratosis, changes in the keratinocytes, in which the cytoplasm contained clear vacuoles, numerous keratohyalin granules and eosinophilic bodies, resulting in a diagnosis of epidermolytic hyperkeratosis of the genitalia. DISCUSSION: Epidermolytic hyperkeratosis of the genitalia is a rare disease, occurring in middle-aged men and women, but chiefly men. The lesions found on the genital organs may be either single, or, more frequently, multiple, and are described as hyperkeratotic papules, which are also reported under the term multiple epidermolytic acanthomas of the genitalia. The aetiology is unknown; certain authors incriminate a traumatic factor; the role of human papillomavirus (HPV) has been suggested but immunohistochemical studies and molecular biology studies generally reveal no viral DNA. Where lesions cause discomfort to the patient, treatment consists of emollients combined with destruction by cryotherapy or CO2 laser. Imiquimod, calcipotriol, tacrolimus and pimecrolimus have all resulted in regression of lesions.

[Health claims for food: a European outlook]. / Allégations de santé des aliments: une perspective communautaire.

The development of health claims for food is driven by a strong industrial impetus and by consumer demand. From the regulatory point of view, the European legislation is fragmented; food and health legislations are contradictory on some aspects. National authorities carry out the regulation of health claims for food according to their own rules. A more comprehensive and harmonized approach within the European Community will be provided by the European regulation currently under discussion between the European Commission and the Parliament. Nevertheless, its final adoption and its implementation will take time. In addition, it won't solve the difficulties related to the assessment of the scientific substantiation of these claims. The role of health authorities in the implementation of rules regarding the safety of consumers remains unadressed.

[Epidemic of Trichophyton tonsurans tinea capitis in a nursery school in the Southern suburbs of Paris]. / Epidémie de teignes à Trichophyton tonsurans dans une école maternelle de la région parisienne.

INTRODUCTION: In March 2001 a school and family survey was conducted in a nursery school in the Southern suburbs of Paris, during an epidemic of Trichophyton tondsurans tinea. PATIENTS AND METHODS: One hundred twenty-nine children aged 3 to 6 were examined as well as 15 adults working in the school. A survey of the contaminated children or asymptomatic carriers was performed. All the children and adults concerned were treated at the same time, without eviction from school. RESULTS: T. tonsurans was detected in 10 cases of tinea (7.7 p.cent of the persons examined), in 18 cases of cutaneous lesions (13.9 p.cent) and in 25 asymptomatic carriers on the scalp (19.4 p.cent). The majority of the positive cases came from the same school class as the original case: 23 of the 26 children (88 p.cent), with 6 tinea, 14 asymptomatic carriers and 13 cutaneous lesions. Only one of the 15 adults exhibited a T. tonsurans cutaneous lesion. Among the 13 families studied, 2 had several members involved, the first being that of the original case (3 tinea and 2 asymptomatic carriers). DISCUSSION: Several important points are underlined by this study: 1) the high contagiousness of T. tonsurans; 2) the detection of 2 mechanisms of indirect contamination (rag doll mascot in the class and the family hair-clipper); 3) the one-year time lapse between the arrival of the contaminating child in the class and the survey, explaining the extent of the contamination; 4) the underestimation of the epidemic due to the lack of mycological examinations; 5) the identification of several dermatophytes in the same school:M. canis, T soudanense and T. tonsurans, and 7) the futility of eviction from school when all the children can be treated.

Spatial analysis and mapping of sexually transmitted diseases to optimise intervention and prevention strategies.

OBJECTIVE: We analysed and mapped the distribution of four reportable sexually transmitted diseases, chlamydial infection/non-gonococcal urethritis (chlamydial infection), gonorrhoea, primary and secondary syphilis (syphilis), and HIV infection, for Wake County, North Carolina, to optimise an intervention. METHODS: We used STD surveillance data reported to Wake County, for the year 2000 to analyse and map STD rates. STD rates were mathematically represented as a spatial random field. We analysed spatial variability by calculating and modelling covariance functions of random field theory. Covariances are useful in assessing spatial patterns of disease locally and at a distance. We combined observed STD rates and appropriate covariance models using a geostatistical method called kriging, to predict STD rates and associated prediction errors for a grid covering Wake County. Final disease estimates were interpolated using a spline with tension and mapped to generate a continuous surface of infection. RESULTS: Lower incidence STDs exhibited larger spatial variability and smaller neighbourhoods of influence than higher incidence STDs. Each reported STD had a clustered spatial distribution with one primary core area of infection. Core areas overlapped for all four STDs. CONCLUSIONS: Spatial heterogeneity within STD suggests that STD specific prevention strategies should not be targeted uniformly across Wake County, but rather to core areas. Overlap of core areas among STDs suggests that intervention and prevention strategies can be combined to target multiple STDs effectively. Geostatistical techniques are objective, population level approaches to spatial analysis and mapping that can be used to visualise disease patterns and identify emerging outbreaks.

An application of the holistochastic human exposure methodology to naturally occurring arsenic in Bangladesh drinking water.

The occurrence of arsenic in drinking water is an issue of considerable interest. In the case of Bangladesh, arsenic concentrations have been closely monitored since the early 1990s through an extensive sampling network. The focus of the present work is methodological. In particular, we propose the application of a holistochastic framework of human exposure to study lifetime population damage due to arsenic exposure across Bangladesh. The Bayesian Maximum Entropy theory is an important component of this framework, which possesses solid theoretical foundations and offers powerful tools to assimilate a variety of knowledge bases (physical, epidemiologic, toxicokinetic, demographic, etc.) and uncertainty sources (soft data, measurement errors, etc.). The holistochastic exposure approach leads to physically meaningful and informative spatial maps of arsenic distribution in Bangladesh drinking water. Global indicators of the adverse health effects on the population are generated, and valuable insight is gained by blending information from different scientific disciplines. The numerical results indicate an increased lifetime bladder cancer probability for the Bangladesh population due to arsenic. The health effect estimates obtained and the associated uncertainty assessments are valuable tools for a broad spectrum of end-users.

Ultrasonic non-destructive testing of pieces of complex geometry with a flexible phased array transducer

Ultrasonic non-destructive testing of components of complex geometry in the nuclear industry faces several difficulties: sensitivity variations due to unmatched contact, inaccurate localization of defects due to variations of transducer orientation, and uncovered area of the component. To improve the performances of such testing and defect characterization, we propose a new concept of ultrasonic contact phased array transducer. The phased array transducer has a flexible radiating surface able to fit the actual surface of the piece to optimize the contact and thus the sensitivity of the test. To control the transmitted field, and therefore to improve the defect characterization, a delay law optimizing algorithm is developed. To assess the capability of such a transducer, the Champ-Sons model, developed at the French Atomic Energy Commission for predicting field radiated by arbitrary transducers into pieces, has to be extended to sources directly in contact with pieces of complex geometry. The good behavior of this new type of probe predicted by computations is experimentally validated with a jointed transducer positioned on pieces of various profiles.

Spatiotemporal analysis of environmental exposure-health effect associations.

The goal of this work is to discuss a general methodology for studying associations between environmental exposures and health effect by means of the spatiotemporal random field theory. This theory is the tool of choice for rigorously accounting for important spatiotemporal variations and uncertainties related to exposures and effect. Within the framework of the random field theory, the Bayesian maximum entropy model neatly synthesizes various sources of physical and epidemiological knowledge into spatiotemporal analysis. Therefore, unlike technical statistics, this approach relies on the blending of substantive physical knowledge with powerful mathematical techniques and a coherent rationale. Given the well-founded fact that certain health effects may be caused by environmental exposures, the significance of these exposures is assessed in terms of a criterion that is based on the joint stochastic representation of exposure and health-effect distributions in space/time. In view of this criterion, the strength and consistency of the exposure-effect association are evaluated on the basis of the health-effect predictions that the combined physico-epidemiologic analysis generates in space/time. The main features of the approach are demonstrated by a simulation example and a real case study involving mortality and cold temperatures in North Carolina. The studies demonstrated the practical usefulness of the stochastic human exposure analysis in assessing the exposure-effect association. The results reported here emphasize the links between spatiotemporal models of physical systems and population health-effect distributions, thus suggesting directions for improving the current understanding of quantitative "exposure-health effect" functions.

A phase I and pharmacokinetic study of melphalan using a 24-hour continuous infusion in patients with advanced malignancies.

The objectives of the present study were to determine the following: (a) the maximum tolerated dose (MTD) of melphalan using a 24-h continuous infusion; (b) the clinical toxicity; and (c) the pharmacokinetic characteristics of melphalan at each dose level. Twenty-one patients with refractory solid tumors were enrolled in the study. Melphalan, packaged in 3% sodium chloride, was administered i.v. over a 24-h period. Patients were assigned to one of three escalating dose levels of melphalan: (a) 20 mg/m2 (n = 5); (b) 30 mg/m2 (n = 7); and (c) 40 mg/m2 (n = 6). Each patient underwent pharmacokinetic evaluation during the first cycle of treatment. Melphalan concentrations in plasma were determined by high-performance liquid chromatography. Toxicity was evaluated after each course of chemotherapy. All of the patients were assessable for toxicity and pharmacokinetics, and 20 patients were assessable for response analysis. A total of 50 courses of melphalan was studied. The MTD was 30 mg/m2. The dose-limiting toxicity was neutropenia and thrombocytopenia. Hematotoxicity was reversible (nadir, 14-15 days; recovery, 3.5 and 12.5 days for 30 and 40 mg/m2, respectively), cumulative, and related to the administered dose and to the history of previous therapy. There were six episodes of neutropenic sepsis. Individual pharmacokinetic parameters were estimated using a Bayesian approach and linear elimination kinetics. Data were compatible with a one-compartment model. Relationships have been found between the area under the plasma concentration-time curve and doses and between Css and doses. Moreover, clearance, t1/2 elimination, and volume of distribution did not change statistically with dose, which suggests linear kinetics. Two partial responses were observed in patients with ovarian carcinoma or adenocarcinoma of unknown primary origin, and another patient had stabilization disease. In conclusion, melphalan MTD was determined to be 30 mg/m2 when administered as a 24-h infusion. Hematological toxicity was the dose-limiting toxicity. The most important nonhematological toxicity encountered was nausea and vomiting. The recommended dose for Phase II studies was 30 mg/m2.

Mutagenesis of the IS1 transposase: importance of a His-Arg-Tyr triad for activity.

Inspection of the primary sequence of the IS1 transposase suggested that it carries residues which are characteristic of the active site of integrases of the bacteriophage lambda family (Int). In particular, these include a highly conserved triad: His-Arg-Tyr. The properties of mutants made at each of these positions were investigated in vivo. The results of several different assays confirm that each is important for transposase activity. Moreover, as in the case of members of the Int family, different mutations of the His residue exhibited different effects. In a particular, His-to-Leu mutation resulted in complete inactivation whereas the equivalent His-to-Gln mutation retained low but significant levels of activity.

DNA splicing by an active site mutant of Flp recombinase. Possible catalytic cooperativity between the inactive protein and its DNA substrate.

Each strand transfer catalyzed by the Flp recombinase is the composite of two transesterification reactions. The active nucleophilic species in the two reactions are the catalytic site tyrosine (Tyr-343) of Flp and the 5'-hydroxyl from the Flp-nicked DNA substrate, respectively. A "half recombination site" is capable of undergoing this pair of transesterifications in the presence of Flp. When the substrate is a half-site containing a chiral phosphorothioate at the exchange point, the Flp reaction yields a product in which the phosphate chirality is retained. A mutant of Flp that lacks the active site tyrosine, Flp(Y343F), is incapable of mediating strand transfer in a full-recombination site but can execute strand transfer in a half-site. The efficiency of this reaction is about 2% of that of the wild type reaction. The activity of Flp(Y343F) is critically dependent on the length of the half-site spacer. Furthermore, in this reaction, the strand cleavage and strand exchange steps cannot be uncoupled. These results strongly suggest a direct attack by the 5'-hydroxyl of the half-site spacer on the phosphodiester at the normal strand transfer point.

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).

Half-site recombinations mediated by yeast site-specific recombinases Flp and R.

The Flp recombinase of Saccharomyces cerevisae and the related R recombinase of Zygosaccharomyces rouxii can efficiently catalyze strand cleavage and strand exchange reactions in half recombination sites. A half-site consists of one recombinase binding element, a recombinase cleavage site on one strand and a 5' spacer hydroxyl group on the other that can initiate the strand exchange reaction. We have studied the various types of strand exchanges that half-sites can participate in. Reaction between a left half-site and a right half-site generates a full recombination site. Strand transfer between two left half-sites or between two right half-sites produces pseudo-full-sites. Strand transfer within a half-site results in a stem-loop or hairpin product. The half-site strand transfer reaction is fairly indifferent to the spacer sequence of the substrate per se and is less sensitive to variations in spacer lengths than a full-site recombination reaction. The optimal spacer length of eight to ten nucleotides observed for the Flp half-site reaction likely permits the most productive catalytic interactions between two Flp monomers bound to each of two partner half-sites. When reacted with a full-site, the half-site can give rise to a normal or reverse recombinant, corresponding to homologous or non-homologous alignments of the spacer sequences during substrate synapsis. The contrary recombination (resulting from non-homologous spacer alignment), whose level is low relative to normal recombination, is partly suppressed when the half-site spacer ends in a 5'-phosphate rather than a 5'-hydroxyl group. Thus, the early steps of recombination, namely synapsis and initial stand transfer, are not dependent on complete spacer homology between the two recombining substrates. The selection of properly aligned substrate partners must occur at the homology dependent branch migration step. In reactions containing a mixture of Flp and R half-sites, Flp and R catalyze strand transfer, almost exclusively, within or between their respective cognate substrates. However, under conditions where self-crosses are inhibited, strand exchange between a Flp half-site and an R half-site appears to be stimulated by a combination of R and Flp.

Half-site strand transfer by step-arrest mutants of yeast site-specific recombinase Flp.

The Flp recombinase of Saccharomyces cerevisae can mediate strand transfer within a half-site, between two half-sites and between a half-site and a full-site. The ability of "step-arrest" mutants of Flp to partake in half-site reactions has been examined. Arg308 variants of Flp, which show little or no strand cleavage in reactions with normal full-sites, execute significant levels of strand transfer in half-site reactions. On the other hand, His305 variants of Flp, which normally accumulate the strand cleavage product from full-sites but do not complete strand transfer, yield only minute amounts of strand transfer products from half-sites. As would be predicted, the step-arrest mutants are unable to produce "normal" or "reverse" recombinants between a half-site and a full-site. The Flp protein is able to form higher-order complexes in association with a half-site. The step-arrest mutants of Flp show specific defects in forming these complexes.

Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan.

The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.

Introduction by site-directed mutagenesis of a tryptophan residue as a fluorescent probe for the folding of Escherichia coli phosphofructokinase.

The leucine residue at position 178 in the major allosteric phosphofructokinase from Escherichia coli has been replaced by a tryptophan using site-directed mutagenesis. Transformation by the mutated gene of pfk- bacteria results into the expression of a pfk+ phenotype and the production of an active enzyme. The modified protein has been purified and its fluorescence properties show that it contains 2 tryptophan residues, the original Trp 311 and the new Trp 178. During unfolding of the protein by guanidine hydrochloride, the changes in the fluorescence of these 2 residues take place at different steps: Trp 311 becomes exposed to solvent when the dimeric form dissociates into monomers, while Trp 178 is exposed only when a folded chain loses its tertiary structure. The mutant enzyme is stabilized by its substrate fructose-6-phosphate against denaturation induced by heat or guanidine hydrochloride.

Role of the C-terminal region in the allosteric properties of Escherichia coli phosphofructokinase-1.

In order to investigate the role of the carboxy-terminal segment in the catalytic, regulatory, and structural properties of the major allosteric phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase: EC 2.7.1.11) from Escherichia coli, the corresponding gene has been modified at either of two sites using oligonucleotide-directed mutagenesis: the codon at position 279 was changed from TAC (Tyr) into TAA (Ochre), and the codon at position 311 from TGG (Trp) into TAG (Amber). The gene mutated at position 279 is not expressed as an active enzyme, probably because a polypeptide chain lacking 41 C-terminal residues cannot fold and/or assemble under the intracellular conditions. The gene mutated at position 311 is expressed as an active enzyme which has been purified to homogeneity. The fluorescence of this protein shows that it has no tryptophan, which confirms that the last nine residues at the carboxy terminal are missing. This derivative has almost the same specific activity and affinities for the two substrates (fructose-6-phosphate and ATP) as intact phosphofructokinase; the saturation by fructose 6-phosphate is also very cooperative. The last nine residues are thus not important for substrate binding, homotropic cooperativity, and catalytic efficiency. The activity of the mutant enzyme is still sensitive to activation by GDP or inhibition by phosphoenolpyruvate, but its affinity for the allosteric effectors is reduced. The carboxy-terminal segment also appears to contribute to the stability of the interactions between subunits: the mutant protein is less stable than the wild type towards denaturation by heat or guanidinium hydrochloride.

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli.

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli. D-mannitol-1-phosphate dehydrogenase was purified to homogeneity from Escherichia coli, and its physicochemical and enzymatic properties were investigated. The molecular weight of the polypeptide chain is 45,000 as determined by polyacrylamide gel electrophoresis in denaturing conditions. High performance size exclusion chromatography gives an apparent molecular weight of 47,000 for the native enzyme, showing that D-mannitol-1-phosphate dehydrogenase is a monomeric NAD-dependent dehydrogenase. D-mannitol-1-phosphate dehydrogenase is rapidly denatured by 6 M guanidine hydrochloride. Non-superimposable transition curves for the loss of activity and the changes in fluorescence suggest the existence of a partially folded inactive intermediate. The protein can be fully renatured after complete unfolding, and the regain of both native fluorescence and activity occurs rapidly within a few seconds at pH 7.5 and 20 degrees C. Such a high rate of reactivation is unusual for a protein of this size. D-mannitol-1-phosphate dehydrogenase is specific for mannitol-1-phosphate (or fructose-6-phosphate) as a substrate and NAD+ (or NADH) as a cofactor. Zinc is not required for the activity. The affinity of D-mannitol-1-phosphate dehydrogenase for the reduced or oxidized form of its substrate or cofactor remains constant with pH. The affinity for NADH is 20-fold higher than for NAD+. The forward and reverse catalytic rate constants of the reaction: mannitol-1-phosphate + NAD+ in equilibrium fructose-6-phosphate + NADH have different pH dependences. The oxidation of mannitol-1-phosphate has an optimum pH of 9.5, while the reduction of fructose-6-phosphate has its maximum rate at pH 7.0. At pH values around neutrality the maximum rate of reduction of fructose-6-phosphate is much higher than that of oxidation of mannitol-1-phosphate. The enzymatic properties of isolated D-mannitol-1-phosphate dehydrogenase are discussed in relation to the role of this enzyme in the intracellular metabolism.