Disseminated Tuberculosis in a Nigerian Adolescent with Linear IgA Bullous Dermatosis: A Case Report and Review of Literature.

Linear IgA bullous dermatosis (LABD) is an auto-immune disease affecting young children and adults, characterized by the linear deposition of IgA at the basement membrane zone with resultant complement activation and a cascade of immune reactions. There is a loss of adhesion at the dermo-epidermal junction and subsequent blister formation. It is a rare disease that has a good prognosis with adequate therapy. However, the underlying depressed immunity associated with the disease may expose them to such infections as tuberculosis. We report the case of an 11-years-old Nigerian female adolescent with LABD, diagnosed at the age of four years but defaulted on follow-up, who developed disseminated tuberculosis (pulmonary, lymph nodes, abdominal and pericardial effusion) seven years after the appearance of the initial blistering skin lesions. She commenced anti-tuberculosis drugs, steroids, and a tube pericardiostomy for the pericardial effusion. Dapsone was initiated for the LABD during the continuation phase of anti-tuberculosis therapy, with subsequent disappearance of the skin rash within two weeks.

La dermatose bulleuse linéaire à IgA (DBL) est une maladie auto-immune affectant les jeunes enfants et les adultes, caractérisée par le dépôt linéaire d'IgA dans la zone de la membrane basale, avec l'activation du complément qui en résulte et une cascade de réactions immunitaires. Il y a une perte d'adhérence à la jonction dermo-épidermique et une formation ultérieure de vésicules. C'est une maladie rare qui a un bon pronostic avec un traitement adéquat. Cependant, l'immunité déprimée sous-jacente associée à la maladie peut les exposer à des infections telles que la tuberculose. Nous rapportons le cas d'une adolescente nigériane de 11 ans atteinte de la LABD, diagnostiquée à l'âge de quatre ans mais en défaut de suivi, qui a développé une tuberculose disséminée (pulmonaire, ganglions lymphatiques, épanchement abdominal et péricardique) sept ans après l'apparition des lésions cutanées vésiculeuses initiales. Elle a commencé à recevoir des médicaments antituberculeux, des stéroïdes et une péricardiostomie par sonde pour l'épanchement péricardique. La dapsone a été initiée pour la DLB pendant la phase de continuation du traitement antituberculeux, avec une disparition de l'éruption cutanée en deux semaines. Mots clés: IgA linéaire, dermatose bulleuse, tuberculose disséminée, adolescent.

Evaluation of emissions and exposures at workplaces using desktop 3-dimensional printer.

There is a paucity of data on additive manufacturing process emissions and personal exposures in real-world workplaces. Hence, we evaluated atmospheres in four workplaces utilizing desktop "3-dimensional" (3-d) printers [fused filament fabrication (FFF) and sheer] for production, prototyping, or research. Airborne particle diameter and number concentration and total volatile organic compound concentrations were measured using real-time instruments. Airborne particles and volatile organic compounds were collected using time-integrated sampling techniques for off-line analysis. Personal exposures for metals and volatile organic compounds were measured in the breathing zone of operators. All 3-d printers that were monitored released ultrafine and fine particles and organic vapors into workplace air. Particle number-based emission rates (#/min) ranged from 9.4 × 109 to 4.4 × 1011 (n = 9samples) for FFF3-d printers and from 1.9 to 3.8 × 109 (n = 2 samples) for a sheer 3-d printer. The large variability in emission rate values reflected variability from the printers as well as differences in printer design, operating conditions, and feedstock materials among printers. A custom-built ventilated enclosure evaluated at one facility was capable of reducing particle number and total organic chemical concentrations by 99.7% and 53.2%, respectively. Carbonyl compounds were detected in room air; however, none were specifically attributed to the 3-d printing process. Personal exposure to metals (aluminum, iron) and 12 different organic chemicals were all below applicable NIOSH Recommended Exposure Limit values, but results are not reflective of all possible exposure scenarios. More research is needed to understand 3-d printer emissions, exposures, and efficacy of engineering controls in occupational settings.

Insights Into Emissions and Exposures From Use of Industrial-Scale Additive Manufacturing Machines.

BACKGROUND: Emerging reports suggest the potential for adverse health effects from exposure to emissions from some additive manufacturing (AM) processes. There is a paucity of real-world data on emissions from AM machines in industrial workplaces and personal exposures among AM operators. METHODS: Airborne particle and organic chemical emissions and personal exposures were characterized using real-time and time-integrated sampling techniques in four manufacturing facilities using industrial-scale material extrusion and material jetting AM processes. RESULTS: Using a condensation nuclei counter, number-based particle emission rates (ERs) (number/min) from material extrusion AM machines ranged from 4.1 × 1010 (Ultem filament) to 2.2 × 1011 [acrylonitrile butadiene styrene and polycarbonate filaments). For these same machines, total volatile organic compound ERs (µg/min) ranged from 1.9 × 104 (acrylonitrile butadiene styrene and polycarbonate) to 9.4 × 104 (Ultem). For the material jetting machines, the number-based particle ER was higher when the lid was open (2.3 × 1010 number/min) than when the lid was closed (1.5-5.5 × 109 number/min); total volatile organic compound ERs were similar regardless of the lid position. Low levels of acetone, benzene, toluene, and m,p-xylene were common to both AM processes. Carbonyl compounds were detected; however, none were specifically attributed to the AM processes. Personal exposures to metals (aluminum and iron) and eight volatile organic compounds were all below National Institute for Occupational Safety and Health (NIOSH)-recommended exposure levels. CONCLUSION: Industrial-scale AM machines using thermoplastics and resins released particles and organic vapors into workplace air. More research is needed to understand factors influencing real-world industrial-scale AM process emissions and exposures.

Particle and vapor emissions from vat polymerization desktop-scale 3-dimensional printers.

Little is known about emissions and exposure potential from vat polymerization additive manufacturing, a process that uses light-activated polymerization of a resin to build an object. Five vat polymerization printers (three stereolithography (SLA) and two digital light processing (DLP) were evaluated individually in a 12.85 m3 chamber. Aerosols (number, size) and total volatile organic compounds (TVOC) were measured using real-time monitors. Carbonyl vapors and particulate matter were collected for offline analysis using impingers and filters, respectively. During printing, particle emission yields (#/g printed) ranged from 1.3 ± 0.3 to 2.8 ± 2.6 x 108 (SLA printers) and from 3.3 ± 1.5 to 9.2 ± 3.0 x 108 (DLP printers). Yields for number of particles with sizes 5.6 to 560 nm (#/g printed) were 0.8 ± 0.1 to 2.1 ± 0.9 x 1010 and from 1.1 ± 0.3 to 4.0 ± 1.2 x 1010 for SLA and DLP printers, respectively. TVOC yield values (µg/g printed) ranged from 161 ± 47 to 322 ± 229 (SLA printers) and from 1281 ± 313 to 1931 ± 234 (DLP printers). Geometric mean mobility particle sizes were 41.1-45.1 nm for SLA printers and 15.3-28.8 nm for DLP printers. Mean particle and TVOC yields were statistically significantly higher and mean particle sizes were significantly smaller for DLP printers compared with SLA printers (p < 0.05). Energy dispersive X-ray analysis of individual particles qualitatively identified potential occupational carcinogens (chromium, nickel) as well as reactive metals implicated in generation of reactive oxygen species (iron, zinc). Lung deposition modeling indicates that about 15-37% of emitted particles would deposit in the pulmonary region (alveoli). Benzaldehyde (1.0-2.3 ppb) and acetone (0.7-18.0 ppb) were quantified in emissions from four of the printers and 4-oxopentanal (0.07 ppb) was detectable in the emissions from one printer. Vat polymerization printers emitted nanoscale particles that contained potential carcinogens, sensitizers, and reactive metals as well as carbonyl compound vapors. Differences in emissions between SLA and DLP printers indicate that the underlying technology is an important factor when considering exposure reduction strategies such as engineering controls.

Tightly locked optical frequency comb from a semiconductor disk laser.

Ultrafast semiconductor disk lasers (SDLs) passively modelocked using semiconductor saturable absorbers mirrors (SESAMs) generate optical frequency combs (OFCs) with gigahertz line spacings - a regime where solid-state and fiber lasers struggle with geometrical and Q-switching limitations. We stabilized both the frequency comb spacing and the offset without any additional external optical amplification or pulse compression. The overall noise performance is competitive with other gigahertz OFCs. A SESAM-modelocked vertical external-cavity surface-emitting laser (VECSEL) at a center wavelength around 1 µm generates 122-fs pulses with 160 mW average output power and we only needed 17-pJ pulse energy coupled into a silicon nitride (Si3N4) waveguide for supercontinuum generation (SCG) and OFC offset stabilization.

Frequency comb offset detection using supercontinuum generation in silicon nitride waveguides.

We present the first direct carrier-envelope-offset (CEO) frequency detection of a modelocked laser based on supercontinuum generation (SCG) in a CMOS-compatible silicon nitride (Si(3)N(4)) waveguide. With a coherent supercontinuum spanning more than 1.5 octaves from visible to beyond telecommunication wavelengths, we achieve self-referencing of SESAM modelocked diode-pumped Yb:CALGO lasers using standard f-to-2f interferometry. We directly obtain without amplification strong CEO beat signals for both a 100-MHz and 1-GHz pulse repetition rate laser. High signal-to-noise ratios (SNR) of > 25 dB and even > 30 dB have been generated with only 30 pJ and 36 pJ of coupled pulse energy from the megahertz and gigahertz laser respectively. We compare these results to self-referencing using a commercial photonic crystal fiber and find that the required peak power for CEO beat detection with a comparable SNR is lowered by more than an order of magnitude when using a Si(3)N(4) waveguide.

Chest pain in asbestos and silica-exposed workers.

BACKGROUND: Chest pain may be the first symptom of developing respiratory malignancy, particularly in subjects with asbestos exposure, yet little information exists on this topic. AIMS: To investigate chest pain in a cohort of subjects exposed to asbestos and silica dust applying for compensation. METHODS: Cross-sectional study using a standardized questionnaire. Data collection included: smoking history, Medical Research Council scales of exercise capacity and respiratory symptoms. RESULTS: We studied 621 subjects. Six disease groups were categorized: asbestosis (n = 27), diffuse pleural thickening (DPT) (132), asbestosis and DPT (14), silicosis (26), pleural plaques only (160) and healthy subjects with a history of dust exposure (256). Crude prevalence rates of chest pain were high, with chest pain approximately twice as common in subjects with asbestos-related disorders and silicosis as in healthy subjects, with an overall frequency of ~40%. However, when other variables were taken into account in a multivariate analysis the differences between groups disappeared. The factor most significantly related to chest pain was age. CONCLUSIONS: Chest pain is apparently common in subjects with asbestos-related disorders and silicosis, but after adjustment for other variables, no increased prevalence was apparent in subjects with pleural disorders. More sophisticated questionnaires and dedicated imaging are required to elucidate this further.

Surveillance of Australian workplace Based Respiratory Events (SABRE) in New South Wales.

BACKGROUND: The Surveillance of Australian workplace Based Respiratory Events (SABRE) New South Wales (NSW) scheme is a voluntary notification scheme established to determine the incidence of occupational lung diseases in NSW Australia. AIMS: Data presented in this paper summarize the last 7 years of reporting to SABRE (June 2001 to December 2008). METHODS: Every 2 months, participating occupational physicians, respiratory physicians and general practitioners (accredited by the NSW WorkCover Authority) reported new cases of occupational lung disease seen in their practices. Data collected include gender, age, causal agent and the occupations and industries believed responsible. Estimated incidence was calculated for each disease. RESULTS: Three thousand six hundred and fifty-four cases were notified to the scheme, consisting of 3856 diagnoses. Most of the cases were males (76%). Pleural plaques [1218 (28%)] were the most frequently reported condition, followed by mesothelioma [919 (24%)]. Silicosis [90 (2%)] and occupational asthma [OA; 89 (2%)] were the most frequently reported non-asbestos-related diseases. Estimated rates for mesothelioma, diffuse pleural thickening (DPT) and OA were 83, 83 and 5 cases per million employed males per year, respectively. Trades such as carpenters and electricians associated with the building industry, electricity supply and asbestos product manufacture were the most common occupations and industries reported. CONCLUSIONS: Asbestos-related diseases are the most frequently reported conditions to SABRE NSW. The very low incidence of OA for NSW most likely reflects under-diagnosis as well as under-reporting. Occupational lung disease is still occurring in NSW despite current preventative strategies. The SABRE scheme currently provides the only available information in this area.

On the origin of selective nitrous oxide N-N bond cleavage by three-coordinate molybdenum(III) complexes.

Reaction of Mo(N[R]Ar)(3) (R = (t)Bu or C(CD(3))(2)CH(3)) with N(2)O gives rise exclusively to a 1:1 mixture of nitride NMo(N[R]Ar)(3) and nitrosyl ONMo(N[R]Ar)(3), rather than the known oxo complex OMo(N[R]Ar)(3) and dinitrogen. Solution calorimetry measurements were used to determine the heat of reaction of Mo(N[R]Ar)(3) with N(2)O and, independently, the heat of reaction of Mo(N[R]Ar)(3) with NO. Derived from the latter measurements is an estimate (155.3 +/- 3.3 kcal.mol(-1)) of the molybdenum-nitrogen bond dissociation enthalpy for the terminal nitrido complex, NMo(N[R]Ar)(3). Comparison of the new calorimetry data with those obtained previously for oxo transfer to Mo(N[R]Ar)(3) shows that the nitrous oxide N-N bond cleavage reaction is under kinetic control. Stopped-flow kinetic measurements revealed the reaction to be first order in both Mo(N[R]Ar)(3) and N(2)O, consistent with a mechanism featuring post-rate-determining dinuclear N-N bond scission, but also consistent with cleavage of the N-N bond at a single metal center in a mechanism requiring the intermediacy of nitric oxide. The new 2-adamantyl-substituted molybdenum complex Mo(N[2-Ad]Ar)(3) was synthesized and found also to split N(2)O, resulting in a 1:1 mixture of nitrosyl and nitride products; the reaction exhibited first-order kinetics and was found to be ca. 6 times slower than that for the tert-butyl-substituted derivative. Discussed in conjunction with studies of the 2-adamantyl derivative Mo(N[2-Ad]Ar)(3) is the role of ligand-imposed steric constraints on small-molecule, e.g. N(2) and N(2)O, activation reactivity. Bradley's chromium complex Cr(N(i)Pr(2))(3) was found to be competitive with Mo(N[R]Ar)(3) for NO binding, while on its own exhibiting no reaction with N(2)O. Competition experiments permitted determination of ratios of second-order rate constants for NO binding by the two molybdenum complexes and the chromium complex. Analysis of the product mixtures resulting from carrying out the N(2)O cleavage reactions with Cr(N(i)Pr(2))(3) present as an in situ NO scavenger rules out as dominant any mechanism involving the intermediacy of NO. Simplest and consistent with all the available data is a post-rate-determining bimetallic N-N scission process. Kinetic funneling of the reaction as indicated is taken to be governed by the properties of nitrous oxide as a ligand, coupled with the azophilic nature of three-coordinate molybdenum(III) complexes.

Synthesis of homochiral tris(2-alkyl-2-aminoethyl)amine derivatives from chiral alpha-amino aldehydes and their application in the synthesis of water soluble chelators.

A novel synthesis of 3-fold symmetric, homochiral tris(2-alkyl-2-aminoethyl)amine (TREN) derivatives is presented. The synthesis is general in scope, starting from readily prepared chiral alpha-amino aldehydes. The optical purity of the N-BOC protected derivatives of tris(2-methyl-2-aminoethyl)amine and tris(2-hydroxymethyl-2-aminoethyl)amine has been ascertained by polarimetry and chiral NMR chemical shift experiments. An X-ray diffraction study of the L-alanine derivative (tris(2-methyl-2-aminoethyl)amine.3 HCl, L-Ala(3)-TREN) is presented: crystals grown from ether diffusion into methanol are cubic, space group P2(1)3 with unit cell dimensions a = 11.4807(2) A, V = 1513.23(4) A(3), and Z = 4. Attachment of the triserine derived backbone tris(2-hydroxymethyl-2-aminoethyl)amine (L-Ser(3)-TREN) to three 3-hydroxy-1-methyl-2(1H)-pyridinonate (3,2-HOPO) moieties, followed by complexation with Gd(III) gives the complex Gd(L-Ser(3)-TREN-Me-3,2-HOPO)(H(2)O)(2), which is more water soluble than the parent Gd(TREN-Me-3,2-HOPO)(H(2)O)(2) and a promising candidate for magnetic resonance imaging (MRI) applications. Crystals of the chiral ferric complex Fe(L-Ser(3)-TREN-Me-3,2-HOPO) grown from ether/methanol are orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions a = 13.6290(2) A, b = 18.6117(3) A, c = 30.6789(3) A, V = 7782.0(2) A(3), and Z = 8. The solution conformation of the ferric complex has been investigated by circular dichroism spectroscopy. The coordination chemistry of this new ligand and its iron(III) and gadolinium(III) complexes has been studied by potentiometric and spectrophotometric methods. Compared to the protonation constants of previously studied polydentate 3,2-HOPO-4-carboxamide ligands, the sum of protonation constants (log beta(014)) of L-Ser(3)-TREN-Me-3,2-HOPO (24.78) is more acidic by 1.13 log units than the parent TREN-Me-3,2-HOPO. The formation constants for the iron(III) and gadolinium(III) complexes have been evaluated by spectrophotometric pH titration to be (log K) 26.3(1) and 17.2(2), respectively.

Slow-binding inhibition of human prostaglandin endoperoxide synthase-2 with darbufelone, an isoform-selective antiinflammatory di-tert-butyl phenol.

The antiinflammatory agent darbufelone, ((Z)-5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl] methylene]-2-imino-4-thiazolidinone, methanesulfonate salt), was discovered as a dual inhibitor of cellular prostaglandin and leukotriene production. To study the mechanism of action of this drug, we expressed human prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2 and purified the recombinant enzymes using buffers that contain octylglucoside. In cyclooxygenase assays following a 15-min incubation of enzyme with inhibitor, darbufelone potently inhibits PGHS-2 (IC(50) = 0.19 microM) but is much less potent with PGHS-1 (IC(50) = 20 microM). Interestingly, when the assay buffer contains traces of Tween 20 (0.0001%), darbufelone appears inactive with PGHS-2 due to a detergent interaction that is detectable by absorption spectroscopy. We therefore used octylglucoside, which does not affect darbufelone in this way, in place of Tween 20 in our PGHS buffers. Inhibition of PGHS-2 with darbufelone is time dependent: with no preincubation, darbufelone is a weak inhibitor (IC(50) = 14 microM), but after a 30-min incubation it is 20-fold more potent. Plots of PGHS-2 activity vs preincubation time at various darbufelone concentrations reach a plateau. This finding is inconsistent with irreversible or one-step slow-binding inhibition. A two-step slow-binding inhibition model is proposed in which the E.I complex (K(i) = 6.2 +/- 1.9 to 14 +/- 1 microM) slowly transforms (k(5) = 0.015-0.030 s(-)(1)) to a tightly bound E.I form with K(i) = 0.63 +/- 0.07 microM and k(6) = 0.0034 s(-)(1). In steady-state kinetics inhibition experiments performed with no preincubation, we find that darbufelone is a noncompetitive inhibitor of PGHS-2 (K(i) = 10 +/- 5 microM). Darbufelone quenches the fluorescence of PGHS-2 at 325 nm (lambda(ex) = 280 nm) with K(d) = 0.98 +/- 0.03 microM. The PGHS substrate, arachidonate, and various cyclooxygenase inhibitors do not alter this binding affinity of darbufelone but a structural analogue of darbufelone competes directly for binding to PGHS-2. Di-tert-butyl phenols such as darbufelone may inhibit PGHS-2 by exploiting a previously unrecognized binding site on the enzyme.

Occupational asthma in adults in six Canadian communities.

We examined the prevalence, population attributable risk (PAR), and clinical characteristics of occupational asthma (OA) in a randomly selected population in six communities in Canada. Our study followed the European Community Respiratory Health Survey protocol. A randomly selected population of 18,701 (87% response rate) persons from the study communities, ranging in age from 20 to 44 yr, completed an initial questionnaire, of whom 2,974 (39% response rate) attended the laboratory and completed supplementary questionnaires. Of these latter individuals, 383 had asthma. Asthma was defined as physician-diagnosed asthma, and adult-onset asthma was defined as a first attack at age 15 yr or older. We used several methods for estimating OA as follows: (1) reporting of a high-risk job (occupation and industry) for OA at the time of asthma onset (Probable OA); (2) reporting of exposure to a substance that may cause OA (Possible OA) while not in a high-risk job at the time of asthma onset; and (3) combination of the PAR for high-risk jobs and exposures. The prevalence (95% confidence interval [CI]) of Probable OA and Possible OA combined was 36.1% (31.3 to 41.0%) among subjects with adult-onset asthma. The occupations most commonly reported in association with OA were nursing in the Probable OA group and clerical and food preparation in the Possible OA group. The clinical characteristics and exposures reported by both groups were similar. The PAR for adult-onset asthma in high-risk jobs and exposures was 18.2%. The assessment of occupation and industry alone, rather than of exposures, may underestimate the contribution of occupational exposures to asthma prevalence.

Spinal nerve segmentation in the chick embryo: analysis of distinct axon-repulsive systems.

In higher vertebrates, the segmental organization of peripheral spinal nerves is established by a repulsive mechanism whereby sensory and motor axons are excluded from the posterior half-somite. A number of candidate axon repellents have been suggested to mediate this barrier to axon growth, including Sema3A, Ephrin-B, and peanut agglutinin (PNA)-binding proteins. We have tested the candidacy of these factors in vitro by examining their contribution to the growth cone collapse-inducing activity of somite-derived protein extracts on sensory, motor, and retinal axons. We find that Sema3A is unlikely to play a role in the segmentation of sensory or motor axons and that Ephrin-B may contribute to motor but not sensory axon segmentation. We also provide evidence that the only candidate molecule(s) that induces the growth cone collapse of both sensory and motor axons binds to PNA and is not Sema3A or Ephrin-B. By grafting primary sensory, motor, and quail retinal neurons into the chick trunk in vivo, we provide further evidence that the posterior half-somite represents a universal barrier to growing axons. Taken together, these results suggest that the mechanisms of peripheral nerve segmentation should be considered in terms of repellent molecules in addition to the identified molecules.

Synthesis of a ligand based upon a new entry into the 3-hydroxy-N-alkyl-2(1H)-pyridinone ring system and thermodynamic evaluation of its gadolinium complex.

The synthesis of a new, more water soluble derivative of TREN-Me-3,2-HOPO (tris[(3-hydroxy-1-methyl-2-oxo-1,2- didehydropyridine-4-carboxamido)ethyl]amine) is presented. The synthesis starts with the condensation reaction of (N-methoxyethylamino)acetonitrile hydrochloride and oxalyl chloride to give 3,5-dichloro-N-(methoxyethyl)-2(1H)-pyrazinone. The 3-position is readily substituted with a benzyloxy group, and the pyrazinone is converted to ethyl 3-(benzyloxy)-N-(methoxyethyl)-2(1H)-pyridinone-4-carboxylate by a Diels-Alder cycloaddition with ethyl propiolate. Basic deprotection of the ester followed by activation, coupling to tren, and acidic deprotection of the benzyl groups gives the ligand TREN-MOE-3,2-HOPO (tris[(3-hydroxy-1-(methoxyethyl)- 2-oxo-1,2-didehydropyridine-4-carboxamido)ethyl]amine). The gadolinium complex of TREN-MOE-3,2-HOPO was prepared by metathesis, starting from gadolinium chloride. The solubility of the new metal complex is significantly enhanced. The four protonation constants (determined by potentiometry) for TREN-MOE-3,2-HOPO (log Ka1 = 8.08, log Ka2 = 6.85, log Ka3 = 5.81, log Ka4 = 4.98) are virtually identical to those reported for the parent ligand. The stability constants for the gadolinium complex of TREN-MOE-3,2-HOPO determined by potentiometry (log beta 110 = 19.69(2), log beta 111 = 22.80(2)) and by spectrophotometry (log beta 110 = 19.80(1), log beta 111 = 22.88(1), log beta 112 = 25.88(1)) differ slightly from those for the parent ligand; this follows from a change in the complexation model in which a new diprotonated species, [Gd(TREN-MOE-3,2-HOPO)(H)2]2+, was included. The presence of this extra species was demonstrated by factor analysis, comparison of spectral data, and nonlinear least-squares refinement. Significant formation of this species is observed between pH 3 and pH 1.5.

A rationalization of the acidic pH dependence for stromelysin-1 (Matrix metalloproteinase-3) catalysis and inhibition.

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His(224). This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu(202) and His(224). The acidic pK(a) 5.6 is attributed to the Glu(202).zinc.H(2)O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Substrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme. substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu(202) and His(224) allows the design of highly selective inhibitors.

Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain.

Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn(2+)- and Ca(2+)-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn(2+) and Ca(2+). While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH(2) with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-alpha converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH(2). Catalytic efficiency with the pro-tumor necrosis factor-alpha converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn(2+)-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.

Neurotoxicity in single photon emission computed tomography brain scans of patients reporting chemical sensitivities.

The subset of patients reporting chemical sensitivity with neurocognitive complaints usually exhibits specific abnormalities of brain metabolism consistent with neurotoxicity, on imaging with single photon emission computed tomography (SPECT). These recurrent neurotoxic patterns are characterized by a mismatch in tracer uptake between early- and late-phase imaging, multiple hot and cold foci throughout the cortex, temporal asymmetry and increased tracer uptake into the soft tissues and, sometimes, the basal ganglia. Previous studies confirm these neurotoxic findings in patients with neurotoxic chemical exposures and breast implants. Affective processes such as depression do not, alone, show this pattern. These abnormalities in SPECT images correlate with documented neurocognitive impairment. Controlled challenges to ambient chemicals can induce profound neurotoxic changes seen on SPECT imaging in chemically sensitive patients. Detoxification treatment techniques frequently produce significant improvement on brain SPECT brain imaging in these patients. Neurotoxicity appears to be characteristic in many cases of chemical sensitivity.

Embryonic lens repels retinal ganglion cell axons.

During development of the vertebrate visual system, retinal ganglion cell (RGC) axons follow a precise path toward their midbrain targets. Although much is known about the cues that direct RGC axons once they have left the optic disc, less is known about the guidance of axons at earlier stages, when RGCs first send out their axons to navigate within the developing retina. Using collagen gel coculture experiments, we find that the embryonic lens produces a powerful diffusible repulsive activity for RGC axons. We also find that this activity is localized to the lens epithelium and not the lens fiber layer, while the pigmented epithelium and vitreous humour are devoid of activity. The further observation that the lens also chemorepels primary sensory axons, but does not repel olfactory bulb axons, shows that this activity is specific for subsets of axons. Our experiments have excluded two candidate repellents for RGC axons (collapsin-1/sema III and chondroitin sulfate proteoglycans). These results implicate the lens in the earliest stages of RGC axon guidance. One function of the lens repellent may be to prevent aberrant targeting toward the lens, and it may also be involved in the directional guidance of RGC axons toward the optic disc.

Pattern formation in a patch occupancy metapopulation model: a cellular automata approach.

The explicit consideration of space in ecological research is of paramount importance to understand the structure and functioning of ecological systems. In this paper we develop a simple spatially explicit metapopulation model in which colonization is constant and independent of the number of occupied patches (i.e. propagule-rain effect, Gotelli, 1991). Extinction, on the other hand, is modelled as a stochastic process whose intensity depends on the number of occupied patches in the neighborhood of each focal patch. Our model is the CA counterpart of two classical patch occupancy metapopulation models. We analytically prove this by showing that our CA converges to the differential equation in the mean-field approximation. The asymptotic behaviour of the system, expressed as the proportion of occupied patches, agrees with the equilibrium proportion of patches derived by using ODEs. In both models, the existence of a rescue-effect increases the range of extinction and colonization parameters over which the system attains complete occupancy of patches. However, in our model this result is strongly influenced by the degree of coupling among patches and is apparent only for local interactions. With local interactions and particular parameter values of colonization and extinction, self-organized spatio-temporal patterns emerge with a fractal-like clustering, even though the environment is spatially homogeneous. Our results point out that the importance of being spatial and discrete (Durrett & Levin, 1994a) in our model is a result of local interactions.

Investigation of a catalytic zinc binding site in Escherichia coli L-threonine dehydrogenase by site-directed mutagenesis of cysteine-38.

L-Threonine dehydrogenase catalyzes the NAD+-dependent oxidation of threonine forming 2-amino-3-ketobutyrate. Chemical modification of Cys-38 of Escherichia coli threonine dehydrogenase, whose residue aligns with the catalytic zinc-binding residue, Cys-46, of related alcohol/polyol dehydrogenases, inactivates the enzyme [B. R. Epperly and E. E. Dekker (1991) J. Biol. Chem. 266, 6086-6092; A. R. Johnson and E. E. Dekker (1996) Protein Sci., 382-390]. To probe its function, Cys-38 was changed to Ser, Asp, and Glu by site-directed mutagenesis. Mutants C38S and C38D were purified to homogeneity and found to be, like the wild-type enzyme, homotetrameric proteins containing one Zn2+ atom per subunit. The circular dichroism spectra of these mutants were essentially identical to that of the wild-type enzyme. Mutant C38S was catalytically inactive but mutant C38D had a specific activity of 0.2 unit/mg, a level approximately 1% that of the wild-type enzyme. After it was incubated with 1 mM Zn2+ and then assayed in the presence of 15 mM Zn2+, mutant C38S showed only a trace of enzymatic activity (i.e., 0.013 unit/mg). Preincubation of mutant C38D with 5 mM Zn2+, Co2+, or Cd2+ increased its activity 57-, 6-, or 3-fold, respectively; 1 mM Mn2+ halved and 0.5 mM Hg2+ abolished activity. Zn2+-stimulated mutant C38D showed these properties: apparent substrate activation at low threonine concentrations, a maximum activity of 27 units/mg with 20 mM threonine, and inhibition by high levels of substrate; an activation Kd = 3 mM Zn2+; and a pH optimum of 8.4 (in contrast to pH 10.3 for the wild-type enzyme). Without added Zn2+, mutant C38D is equally active with threonine and 2-amino-3-hydroxypentanoate, but Zn2+-activated mutant C38D is 10-fold more reactive with threonine than with 2-amino-3-hydroxypentanoate. In the absence of added metal ions, wild-type enzyme similarly uses substrates other than threonine and shows a dramatic increase in activity with only threonine when stimulated by either Cd2+ or Mn2+; added Zn2+ has no effect on activity with threonine. Cys-38 of threonine dehydrogenase, therefore, is located in an activating divalent metal ion-binding site. Having a negatively charged residue like Asp in this position allows the binding of a catalytic Zn2+ ion which enhances activity with threonine and reduces activity with substrate analogs. Whether Cys-38 of wild-type threonine dehydrogenase binds a catalytic metal ion (possibly Zn2+) in vivo remains to be established.