X-ray structure of a lectin-bound DNA duplex containing an unnatural phenanthrenyl pair.

DNA duplexes containing unnatural base-pair surrogates are attractive biomolecular nanomaterials with potentially beneficial photophysical or electronic properties. Herein we report the first X-ray structure of a duplex containing a phen-pair in the center of the double helix in a zipper like stacking arrangement.

What we have learned from crystal structures of proteins to receptor function.

The activity of ligand gated channels is crucial for proper brain function and dysfunction of a single receptor subtype have led to neurological impairments ranging from benign to major diseases such as epilepsy, startle diseases, etc. Molecular biology and crystallography allowed the characterization at the atomic scale of the first four transmembrane ligand gated channels and of proteins sharing a high degree of homology with the neurotransmitter-binding domain. Gaining an adequate knowledge of the structural features of the ligand binding pocket led to the possibilities of developing virtual screening based approaches and probing in silico the docking of very large numbers of molecules. Development of new computing tools further extended such possibilities and rendered possible the screening of the chemical universe database GDB-11, which contains all possible organic molecules up to 11 atoms of C, N, O and F. In the case of the nicotinic acetylcholine receptors molecules identified using such screening methods were synthesized and characterized in binding assays and their pose determined in crystal structure with the acetylcholine binding protein. However, in spite of these thorough approaches, functional studies revealed that these molecules had a greater affinity for the pore domain of the channel and acted as open channel blocker rather than binding site antagonist. In this work, we discuss the potential and current limitations of how progresses made with the crystal structures of ligand gated channels, or ligand binding proteins, can be used in combination with virtual screening and functional assays, to identify novel compounds.

[Febrile cellulitis surrounding a scar revealing a large immunoblastic B-cell lymphoma]. / Dermohypodermite fébrile péricicatricielle révélatrice d'un lymphome B immunoblastique à grandes cellules.

BACKGROUND: Secondary skin sites of lymphoma appear in the advanced stages of the disease. We report the first case of a pericicatricial skin infiltration, mimicking febrile dermohypodermitis, revealing diffuse immunoblastic large B-cell non-Hodgkin's lymphoma. PATIENTS AND METHODS: Four months after decompressive cervical laminectomy, a 56-year-old man presented an inflammatory pericicatricial patch evoking cellulitis in a setting of hyperthermia and lymphadenopathy. Blood cultures and bacteriological analysis of skin biopsy samples were negative. The images showed infiltration of the soft subcutaneous areas and polyadenopathy. Two weeks later, several subcutaneous nodules appeared on the trunk. Histological analysis and immunolabelling pointed to immunoblastic large B-cell non-Hodgkin's lymphoma. A clone of B lymphocytes CD45+, CD20+, CD79a+, Bcl2+, CD5+, MUM1+, CD3-, CD10-, CD23- and Bcl6- was seen. The remainder of the extension examination was negative. CHOP-rituximab polychemotherapy resulted in complete regression of all lesions, notably the inflammatory cervical plaque. DISCUSSION: Secondary skin manifestations of lymphoma are generally non-specific (pruritus, ichthyosis, purpura, etc.) rather than specific in terms of lymphoid infiltration. As in our patient, certain cutaneous sites of lymphoma may have a misleading clinical presentation, histological analysis alone was able to provide a conclusive diagnosis. In our patient, the highly specific infiltration seen around the entire scar could either suggest a Koebner phenomenon or point to a role of the cutaneous aggression within the development of an inflammatory process contributing to pericicatricial infiltration by lymphoid cells. Locoregional invasion from the osseous part of the cervical spine and not macroscopically diagnosed during neurosurgery could also be responsible.

[Syringotropic and pilotropic cutaneous T-cell lymphoma without follicular mucinosis]. / Mycosis fongoïde syringotrope et pilotrope sans mucinose folliculaire.

BACKGROUND: Mycosis fongoides rarely exhibits predilection for infiltration of hair follicles and eccrine glands. We report the case of a patient with cutaneous T-cell lymphoma with syringotropism and pilotropism without follicular mucinosis. CASE REPORT: A 51-year-old man presented with erythematous infiltrated patches with alopecia and anhydrosis, cystic lesions, comedon-like lesions, follicular keratosis and an ulcer over the lower left leg. Clinical examination revealed no palpable adenopathy or hepatosplenomegaly. The patient complained about hypohydrosis. Histological examination of skin biopsies evidenced pilotropic cutaneous T-cell lymphoma without follicular mucinosis. Immunohistological examination and T-cell receptor B-chain gene rearrangement analysis showed a clonal population of T-cells. Moreover sweat glands and sweat ducts were infiltrated by atypical lymphocytes with syringolymphoid hyperplasia. DISCUSSION: Syringolymphoid hyperplasia and folliculotropism without follicular mucinosis are rarely seen in mycosis fongoides. Deep biopsies of adnexal structures are required for this critical diagnosis and clonal rearrangement of TCR genes is a reliable means of assessing clonality. Syringolymphoid hyperplasia and pilotropism without mucinosis are variants of cutaneous T-cell lymphomas.

Isolation and biochemical characterization of two novel metagenome-derived esterases.

The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to beta-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50 degrees C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47 degrees C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.

[Acrodermatitis enteropathica-like syndrome secondary to branched-chain amino acid deficiency during treatment of maple syrup urine disease]. / Pseudo-acrodermatite entéropathique secondaire à un déficit en acides aminés ramifiés au cours du traitement d'une leucinose.

INTRODUCTION: Clinical pictures resembling acrodermatitis enteropathica have been described in acquired zinc deficiency and deficiencies of other nutrients such as biotin, essential fatty acids and amino acids as well as biotin metabolism disorders. We describe the case of an infant with maple syrup urine disease who developed an acrodermatitis-like syndrome due to iatrogenic valine and isoleucine deficiency. CASE-REPORT: A diagnosis of maple syrup urine disease was made in a 5-month-old infant girl with severe neurologic disorders with extremely high levels of the three branched-chain amino acids (leucine, valine and isoleucine) in plasma and urine. Seven days after the start of therapy with a diet excluding these branched-chain amino acids, plasma isoleucine and valine concentrations were low while plasma leucine remained elevated. At the same time, a periorificial and acral dermatitis appeared together with diarrhea. Serum zinc concentrations were normal. A diagnosis of acrodermatitis enteropathica-like syndrome secondary to isoleucine and valine deficiency was suspected. Valine and isoleucine supplementation resulted in rapid resolution of the eruption. DISCUSSION: Several cases of acrodermatitis enteropathica-like eruptions resulting from therapeutic protein restriction diets have been described in infants with different aminoacidopathies. The accompanying dermatosis was associated with a raised plasma leucine/isoleucine ratio and/or isoleucine deficiency, or valine deficiency. While the exact pathogenesis of the skin lesions has not been established, these observations show that branched-chain amino acids are essential for normal growth and differentiation of keratinocytes. The essential role of isoleucine is further substantiated by the fact that its presence is critical in keratinocyte culture media, with growth arrest occurring upon its depletion.

[Fluindione-induced acute exanthematous pustulosis with renal involvement]. / Pustulose exanthématique aiguë généralisée à la fluindione avec atteinte rénale.

INTRODUCTION: Fluindione (Previscan) is an oral anti-vitamin K anticoagulant, widely prescribed in France. Contrary to phenindione, which is also an indanedione derivative, very few cases of immunoallergic reactions have been described. CASE REPORT: A 68 year-old man, treated with fluindione for cardiac arrhythmia, presented with a pustular eruption and erythema twenty days after initiation of treatment. The eruption was associated with hyperthermia, arthralgia, neutrophilia (11,000/mm2), hepatic cytolysis and renal involvement including acute renal failure, hematuria and proteinuria. In view of the absence of any earlier case in the literature, we did not impute fluindione and the drug was reintroduced and led to the rapid recurrence of all the same manifestations. DISCUSSION: These manifestations were consistent with an immunoallergic reaction to fluindione (probable intrinsic imputability I3) and acute interstitial nephritis (probable intrinsic imputability I3). We believe this is the first case of acute generalized exanthematous pustulosis induced by fluindione (intrinsic imputability Bo). A few rare cases of fluindione-induced hypersensitivity reactions and acute interstitial nephritis, however, have been described.

Novel methods for biocatalyst screening.

There have been a number of recent advances in catalysis assays applicable for screening biocatalyst libraries in high-throughput format. These include instrumental assays such as high-performance liquid chromatography, mass spectrometry, capillary electrophoresis and IR-thermography, reagent-based assays producing spectroscopic signals (UV/VIS or fluorescence) in response to reaction progress, and assays based on fluorogenic or chromogenic substrates. These fluorogenic substrates enable the assaying of a variety of enzymes in enantioselective and stereoselective manner, including alcohol dehydrogenases, aldolases, lipases, amidases, epoxide hydrolases and phosphatases.

Pivalase catalytic antibodies: towards abzymatic activation of prodrugs.

Screening of monoclonal-antibody libraries generated against the tert-butyl phosphonate hapten 2 and the chloromethyl phosphonate hapten 3 with pivaloyloxymethyl-umbelliferone 1 as a fluorogenic substrate led to the isolation of eleven catalytic antibodies with rate accelerations around kcat/ kuncat = 10(3). The antibodies are not inhibited by the product and accept different acyloxymethyl derivatives of acidic phenols as substrates. The highest activity was found for the bulky, chemically less-reactive pivaloyloxymethyl group: there is no activity with acetoxymethyl or acetyl esters. This difference might reflect the preference of the immune system for hydrophobic interactions in binding and catalysis. Pivalase catalytic antibodies might be useful for activating orally available pivaloyloxymethyl prodrugs.

High-throughput screening for biocatalysts.

Recent progress in high-throughput enzyme assays includes new examples of fluorogenic and chromogenic substrates, fluorescence resonance energy transfer substrates, and applications of the pH and pM indicator methods. Recent developments of Horeau's pseudo-enantiomer derivatisation method to screen enantioselectivities in high-throughput have also been reported.

Fluorogenic polypropionate fragments for detecting stereoselective aldolases.

A series of fluorogenic polypropionate fragments has been prepared. These undergo retroaldolization to an intermediate aldehyde that liberates the fluorescent product umbelliferone by a secondary beta-elimination reaction. leading to a >20-fold increase in fluorescence (lambda(em) = 460 +/- 20 nm, lambdaex = 360 +/- 20 nm). By applying the principle of microscopic reversibility to the reversible aldol reaction, we can use these substrates to detect stereoselective aldolases. Test substrates are available to probe the classical cases of syn- and anti-selective aldolization (11a-d), Cram/ anti-Cram-selective aldolization (10a-d), and double stereoselective aldolization (3a-h). The selectivity of aldolase antibody 38C2 for these substrates is demonstrated as an example. The assay is suitable for high-throughput screening for catalysis in microtiter plates, and therefore provides a convenient tool for the isolation of new stereoselective aldolases from catalyst libraries.

Stereoselective inhibition of alpha-L-fucosidases by N-benzyl aminocyclopentitols.

[structure: see text] (1R,2R,3R,4R,5R)-4-Amino-5-methylcyclopentane-1,2,3 -tr iol 8, its 4S stereoisomer 9, and their acyclic analogues (R)- and (S)-2-aminobutanol 11 and 12 are selective but moderate inhibitors of alpha-L-fucosidases. N-Benzylation selectively enhances inhibition potency for aminocyclopentitol 8 (--> 1, K(i) = 6.8 x 10(-)(7) M) but decreases inhibition for its 4S-stereoisomer 9 (--> 2, K(i) = 1.1 x 10(-)(4) M) and for the aminobutanols 11 (--> 13, no inhibition) and 12 (--> 14, no inhibition).

Synthesis and evaluation of aminocyclopentitol inhibitors of beta-glucosidases.

[reaction: see text] (1R,2S,3S,4R,5R)-4-Amino-5-(hydroxymethyl)cyclopentane-1,2,3-triol 1, prepared from D-glucose, inhibits beta-glucosidases from Caldocellum saccharolyticum (Ki = 1.8 x 10(-7) M) and from almonds (Ki = 3.4 x 10(-6) M). Inhibition is not influenced by N-ethylation (--> 15) but is strongly reduced upon N-acetylation (--> 12). Inversion of stereochemistry at C(5) (--> 14) has little effect on inhibition of beta-glucosidases. These experiments suggest that 1 acts as an analogue of a protonated beta-glucoside.

Phosphofurylalanine, a stable analog of phosphohistidine.

Phosphorylated histidine residues occur in a number of signal-transduction pathways in bacteria as well as in eukaryotes. Phosphohistidine is hydrolytically labile and therefore difficult to study, this by contrast to stable phosphoserine, phosphothreonine or phosphotyrosine. Here we report the design and enantioselective synthesis of (4'-phospho-2'-furyl)alanine 1, a non-hydrolyzable analog of 1-phosphohistidine. This novel amino-acid should be useful to synthesize peptides incorporating a stable analog phosphohistidine.

[Sporadic superficial pemphigus in the child: 2 cases]. / Pemphigus superficiel sporadique de l'enfant: 2 observations.

INTRODUCTION: Excepting the endemic foliaceus form, childhood pemphigus is uncommon. We report two cases of pemphigus foliaceus in children with typical clinical manifestations. CASE REPORTS: Case n(o) 1. A 5-year-old girl was seen for a vesiculobullous crusted dermatosis involving the trunk and the face which had developed over the last 5 months, predominantly in periorificial and fold localizations. Histology showed intragranulous acatholysis. Direct skin immunofluorescence was positive for anti-intercellular substance IgG and C3. Indirect immunofluorescence was positive for anti-intercellular substance antibodies at 1/500. The diagnosis of superficial pemphigus was retained and the child was given dapsone associated with systemic prednisone (1.5 then 2.5 mg/kg/d). Dapsone was stopped on day 15 due to poor hematological tolerance. Outcome was favorable allowing withdrawal of prednisone at 18 months. Case n(o) 2. A 6-year-old had developed since the age of 18 months a generalized and polycyclic pruriginous erythemato-squamous dermatosis with oozing discharge which started and predominated on the face (periorificial zones). Trace element (copper, selenium, zinc) and vitamin (A, E and B1) assays were within the normal range. Glucagon was normal. Histological examinations of several biopsies were non-contributive. Diagnosis of pemphigus foliaceus was finally obtained after repeated direct immunofluorescence tests which revealed anti-intercellular substance IgG. Indirect immunofluorescence was negative. The child was given prednisone (2 mg/kg/d). DISCUSSION: In children, pemphigus foliaceus has an exceptional frequency and diagnosis is often made quite late (mean 8 months). The diagnosis should always be entertained in children who develop chronic extensive erythemato-squamous and crusted dermatosis, even if formation is absent. Direct skin immunofluorescence confirms the diagnosis and should be repeated if negative in cases with highly suggestive clinical presentations. It would be reasonable to attempt "minor" treatments as the first line approach. Systemic corticosteroids are however the treatment of choice despite the risk of classical side effects. Childhood pemphigus foliaceus is not an attenuated clinical form of adult pemphigus. Mortality is not negligible and is close to that in adults.

Anomer-selective inhibition of glycosidases using aminocyclopentanols.

[reaction: see text] (1S,2S,3S,4R,5R)-4-amino-5-(hydroxymethyl)cyclopentane-1,2,3-triol 1 is prepared stereoselectively from D-lyxose and displays anomer-selective inhibition for beta-galactosidase (Ki = 3.0 x 10(-6) M) and beta-glucosidase (Ki = 1.5 x 10(-7) M), over alpha-galactosidase (Ki = 2.3 x 10(-5) M) and alpha-glucosidase (IC50 = 1.0 x 10(-4) M). There is no observable cross-reactivity with alpha-mannosidase, beta-mannosidase, or alpha-L-fucosidase.

An enantioselective fluorimetric assay for alcohol dehydrogenases using albumin-catalyzed beta-elimination of umbelliferone.

3-hydroxybutyl umbelliferyl ethers (R)-1 and (S)-1 are fluorogenic substrates for alcohol dehydrogenases. Their oxidation forms ketone 2, which undergoes beta-elimination of umbelliferone under catalysis by bovine serum albumin, leading to a > 20-fold fluorescence increase at lambda em = 460 +/- 20 nm (lambda ex = 360 +/- 20 nm). Enantioselectivity is determined in two separate tests with each enantiomeric substrate.

Asymmetric organic synthesis with catalytic antibodies.

The science of catalytic antibodies has undergone a rapid maturation process within its first nine years of existence. From initial 'proof of concept' and demonstration of fundamental, enzyme-like characteristics, antibodies have been shown to catalyze a remarkably broad scope of organic transformations, including difficult and unfavorable chemical reactions. Yet, the ultimate testing ground for new concepts in organic chemistry has always been the synthesis of natural products. Here we focus on several issues related to the applicability of antibody catalysis in organic synthesis. We show that (a) in the hydrophobic environment of the antibody active site, short-lived intermediates can be formed and reacted in a controlled way, thus allowing antibodies to catalyze reactions that are normally incompatible with aqueous media, (b) the intrinsic order of reactivity (chemoselectivity) in a series of structurally related enol ethers and ketals can be inverted from 1:10 in the uncatalyzed hydrolysis reaction to 1000:1 under antibody catalysis, and (c) an efficient total synthesis of alpha-multistriatin, an important, biologically active natural product can be achieved via antibody catalysis.

A general assay for antibody catalysis using acridone as a fluorescent tag.

A simple and highly sensitive catalysis assay is demonstrated based on analyzing reactions with acridonetagged compounds by thin-layer chromatography. As little as 1 pmol of product is readily visualized by its blue fluorescence under UV illumination and identified by its retention factor (Rf). Each assay requires only 10 microliters of solution. The method is reliable, inexpensive, versatile, and immediately applicable in repetitive format for screening catalytic antibody libraries. Three examples are presented: (i) the epoxidation of acridone labeled (S)-citronellol. The pair of stereoisomeric epoxides formed is resolved on the plate, which provides a direct selection method for enantioselective epoxidation catalysts. (ii) Oxidation of acridone-labeled 1-hexanol to 1-hexanal. The activity of horse liver alcohol dehydrogenase is detected. (iii) Indirect product labeling of released aldehyde groups by hydrazone formation with an acridone-labeled hydrazide. Activity of catalytic antibodies for hydrolysis of enol ethers is detected.

Antibody catalysis of a reaction otherwise strongly disfavoured in water.

Several examples have been reported recently of antibody catalysis of reactions that are strongly disfavoured because of the high free energy of the transition state. Here we show that catalytic antibodies can be used to promote a particularly useful kind of reaction from a synthetic point of view: one involving an intermediate that is highly unstable in water. We show that an antibody elicited against the quaternary ammonium ion 4a (Fig. 1) catalyses the protonation of the enol ether 1 to form, with complete enantioselectivity, an oxocarbonium intermediate. This species is highly reactive in water, and would normally react with a water molecule to give the corresponding ketone 2. But the antibody provides a hydrophobic environment that allows the oxocarbonium ion instead to undergo an intramolecular reaction to form an enantiomerically pure ketal 3. This result shows that catalytic antibodies can exclude solvent molecules entirely from crucial steps on the reaction pathway.