Interleukin-1A +4845(G> T) polymorphism is a factor predisposing to acne vulgaris.

Acne vulgaris is a common chronic inflammatory skin disease of multifactorial origin. The aim of this study was to clarify whether known polymorphisms of the interleukin-1A (IL1A) and IL1RN genes play a role in the pathogenesis of acne vulgaris. A positive association was found between the minor T allele of the IL1A +4845(G>T) single nucleotide polymorphism (SNP) and acne, whereas no association was found with respect to any alleles of the variable number of tandem repeats (VNTR) polymorphism of the IL1RN gene. The severity of inflammatory acne symptoms correlated with the percentage of individuals carrying the homozygote T/T genotype. These results may help to elucidate the molecular events leading to the development of acne.

Quantitative characterization of regional differences in the GABAA-receptor alpha1-subunit mRNA expression in the rat brain.

Inhibition in the central nervous system is largely mediated by local-circuit neurons that release GABA (gamma-amino-butyric acid). GABAA-receptors play a major role in virtually all brain physiological functions and serve as targets for numerous classes of drugs, used both in clinical practice and as research tools. These receptors are heteropentamers, alpha1 being the most widely occurring subunit; therefore it is the best candidate to be studied in pathological conditions where the inhibitory system might be altered (e.g. epilepsy). We compared quantitatively the regional distribution of GABAA-receptor alpha1-subunit (GABAAR-alpha1) expression in three brain areas: neocortex, hippocampus and cerebellum by RT-qPCR. TaqMan probe was used in order to avoid detection of non-specific amplification products and synaptophysin as internal control. This substance was chosen because it has a stable expression restricted to neurons, and contrary to GAPDH, the most commonly used reference gene for expression analysis, synaptophysin expression is not modified in animal models of epilepsy. Expression of synaptophysin was higher than expression of GABAAR-alpha1 in all samples from the central nervous system. The latter was significantly different among the studied brain areas. It was the smallest in the hippocampus, intermediate in the neocortex and the highest in the cerebellum. Interanimal differences were small for any brain region under study. These results indicate that combination of TaqMan real-time PCR method with synaptophysin as internal control can reliably measure the relative expression of GABAAR-alpha1 mRNA, and are suitable for investigating the modifications that appear under pathological conditions and/or diverse experimental paradigms.

Budesonide, but not tacrolimus, affects the immune functions of normal human keratinocytes.

Topical immunosuppressant therapy is widely used in the treatment of inflammatory skin diseases such as psoriasis and atopic dermatitis. Besides its beneficial therapeutic effects, application of topical anti-inflammatory drugs may render the epidermis more vulnerable to invading pathogens by suppressing innate immune responses in keratinocytes, such as cytokine production and Toll-like receptor (TLR) expression. In order to evaluate and compare the immunosuppressive effects of different immunosuppressant drugs on keratinocytes, we treated lipopolysaccharide (LPS)-stimulated and -unstimulated normal human keratinocytes with the synthetic corticosteroid budesonide and the macrolide tacrolimus. The expressions of the pattern recognition receptors (PRRs) TLR2 and TLR4 were measured by quantitative RT-PCR, pro-inflammatory cytokines IL-1alpha, IL-8 and TNF-alpha were monitored by quantitative RT-PCR and by ELISA, and alterations in TLR2 protein level were measured by flow cytometry. Budesonide had a suppressive effect on both constitutive and LPS-induced IL-8 gene expression. The amount of TNF-alpha mRNA was diminished in unstimulated keratinocytes, while TLR2 mRNA expression was markedly enhanced both in unstimulated and LPS-treated cells after incubation with budesonide. This increase in TLR2 mRNA expression was also detectable at the protein level in LPS-stimulated cells. Tacrolimus had no effect on any of the examined genes. Budesonide, but not tacrolimus, significantly inhibited the NF-kappaB-dependent luciferase reporter activity in HaCaT cells after induction with LPS or TNF-alpha. Although tacrolimus and budesonide are both effective treatments in some inflammatory skin diseases, the data provided here imply differences in local therapeutic and adverse effects of these two topical immunosuppressants.

The non-mevalonate pathway of isoprenoids: genes, enzymes and intermediates.

Although the mevalonate pathway had been considered for a long time as the unique source of biosynthetic isoprenoids, an alternative pathway has recently been discovered. The first intermediate, 1-deoxy-D-xylulose 5-phosphate, is assembled by condensation of glyceraldehyde 3-phosphate and pyruvate. A skeletal rearrangement coupled with a reduction step affords the branched-chain polyol, 2C-methyl-D-erythritol 4-phosphate, which is subsequently converted into a cyclic 2,4-diphosphate by the consecutive action of three enzymes via nucleotide diphosphate intermediates. The genes specifying these enzymes have been cloned from bacteria, plants and protozoa. Their expression in recombinant bacterial hosts has opened the way to the identification of several novel pathway intermediates.

A comparison of the effects of ifosfamide vs. mafosfamide treatment on intracellular glutathione levels and immunological functions of immunocompetent lymphocyte subsets.

This study compares the effects of ifosfamide treatment with those of mafosfamide treatment with respect to important immunological functions and intracellular glutathione (GSH) levels of immunocompetent lymphocyte subsets such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The proliferative and cytotoxic capacity of human peripheral blood lymphocyte (PBL) subsets was measured by a standard [3H] thymidine uptake assay and a [51Cr] release assay; the intracellular glutathione levels were determined by using an established HPLC method described by Reed. Following incubation of human PBLs with the activated forms of ifosfamide (4-OH-IF) and mafosfamide (4-OH-CP), the proliferative capacity of recombinant interleukin 2 (rIL-2)-stimulated PBLs was reduced by both drugs in a dose-dependent manner. However, a three-fold higher concentration of ifosfamide compared with mafosfamide is needed to achieve a comparable inhibition rate in the proliferative capacity of theses lymphocytes. Separation of PBLs into a CD3+ CTL and a CD3- NK subpopulation revealed that proliferative activity was reduced in both subpopulations in a dose-dependent manner by ifosfamide and mafosfamide. However, growth inhibition was much more pronounced in the CD3+ CTL compared with the CD3- NK cells. The intracellular GSH level in CTL, and to a lower extent in NK cells, was reduced more substantially following an ifosfamide treatment compared with a mafosfamide treatment. With respect to our previous finding that an ifosfamide-induced reduction of intracellular GSH levels correlates with decreased cytotoxic function, in this study we compared the effects of ifosfamide treatment with those of mafosfamide treatment on the cytolytic activity of lymphocyte subpopulations. The cytotoxic activity of CD3+ CTL against allogeneic target cells (B-lymphoblastoid cells) was significantly reduced after preincubation with either activated ifosfamide or mafosfamide. In contrast, the lysis of NK-sensitive tumor target cells (K562), mediated by CD3- NK cells is only affected if the effector cells are exposed to high concentrations (100 microM) of activated ifosfamide. The cytotoxic activity of NK cells pretreated with high concentrations of activated mafosfamide (33 microM) had no significant inhibitory effect on the cytotoxic function. Taken together, our findings were as follows: 1) A three-fold higher concentration of activated ifosfamide compared with mafosfamide results in a comparable inhibition of the proliferative activity, in vitro. 2) The intracellular GSH levels of unseparated rIL-2 activated lymphocytes were reduced by ifosfamide and mafosfamide at concentrations above 16 microM. 3) Separated NK cells compared with CTLs are more resistant to treatment with ifosfamide with respect to their intracellular GSH levels. This phenomenon is even more pronounced after treatment with mafosfamide. 4) The reduction in intracellular GSH levels after treatment with ifosfamide and mafosfamide could be correlated with a reduction in the cytotoxic activity.

Biosynthesis of terpenoids: 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Escherichia coli is a class B dehydrogenase.

1-Deoxy-D-xylulose-5-phosphate is converted into 2-C-methyl-D-erythritol-4-phosphate by the catalytic action of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr protein) using NADPH as cofactor. The stereochemical features of this reaction were investigated in in vitro experiments with the recombinant Dxr protein of Escherichia coli using (4R)- or (4S)-[4-(2)H(1)]NADPH as coenzyme. The enzymatically formed 2-C-methyl-D-erythritol-4-phosphate was isolated and converted into 1,2:3,4-di-O-isopropylidene-2-C-methyl-D-erythritol; NMR spectroscopic investigation of this derivative indicated that only (4S)-[4-(2)H(1)]NADPH affords 2-C-methyl-D-erythritol-4-phosphate labelled exclusively in the H(Re) position of C-1. Stereospecific transfer of H(Si) from C-4 of the cofactor identifies the Dxr protein of E. coli as a class B dehydrogenase.

Morphological identification of neuron types in the rat hippocampus.

The cerebral cortex ensures an optimal interaction of mammals, including humans, with their environment, by encoding, storing and combining information about the surrounding world and the internal milieu. Probably the simplest and the most popular region for studying the cortical network is the hippocampal CA1 area, because it has the least heterogeneous neuronal population, the somata and dendrites of principal neurons (pyramidal cells) are arranged into well defined layers and the extrinsic and intrinsic inputs are segregated. The relatively homogeneous pyramidal cell population is supported by a very heterogeneous GABAergic interneuron population, which provides not only general inhibition, but also regulates the precise timing of pyramidal cell activity. Interneurons usually innervate distinct domains of the surface of their target cell. The strategic placement of inhibitory synapses, indicate that GABAergic interneurons belonging to different classes serve distinct functions in the hippocampal network. Neuron types are usually defined according to various morphological, molecular and physiological features. Under typical experimental conditions only some of these parameters are available, therefore an important scientific question is: which partial measures are sufficient for correct recognition of a class of cell. By immunohistochemistry it is possible to stain all neurochemically identical neurons in a given brain region, therefore it is the most widely used method for identifying neuron classes. This review presents the neuron types identified so far in the area CA1 of the rat hippocampus with special emphasis on the immunocytochemical characterization of these cells.

Endogenous phospholipid metabolite containing topical product inhibits ultraviolet light-induced inflammation and DNA damage in human skin.

BACKGROUND: N-palmitoylethanolamine (PEA) and organic osmolytes are endogenous components of the human epidermis and are generated from phospholipids in the stratum granulosum. PEA has been shown to exert potent antioxidant and anti-inflammatory activities. The endogenous organic osmolytes such as betaine and sarcosine control skin humidity, but have also been shown to inhibit ultraviolet (UV) light-induced oxidative stress in keratinocytes. OBJECTIVES: To investigate the effect of a PEA- and organic osmolyte-containing topical product (Physiogel AI) on the development of UV light-induced erythema, thymine dimer formation and p53 tumor suppressor gene activation, as well as intercellular adhesion molecule 1 (ICAM-1) and Ki67 expression in normal human skin. METHODS: The UV-induced erythema was measured by a spectrofluorometric method. Thymine dimers, p53, ICAM-1 and Ki67 were detected in skin biopsies using immunohistochemistry. RESULTS: Physiogel AI cream significantly inhibited the development of UV light-induced erythema and thymine dimer formation in normal human skin, but did not alter the number of Ki67+ proliferating keratinocytes and the expression of p53 and ICAM-1. CONCLUSIONS: Our results suggest that PEA and organic osmolytes might represent a new generation of compounds which suppress UV-induced photodamage.

Substrate channeling in the lumazine synthase/riboflavin synthase complex of Bacillus subtilis.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of three alpha subunits. The beta subunits catalyze the condensation of 5-amino-6-ribityl-amino-2,4(1H,3H)-pyrimidinedione (PYR) with 3,4-dihydroxy-2-butanone 4-phosphate (DHB) yielding 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation. The second product of this reaction is PYR, which is also a substrate of the beta subunits and can be recycled in the catalytic process. Sigmoidal kinetics would be expected for the formation of riboflavin from PYR and DHB and are indeed observed with mixtures of artifactual beta 60 capsids and alpha subunit trimers. In contrast, the formation of riboflavin from PYR and DHB by the native alpha 3 beta 60 is characterized by a finite initial rate, which is similar to the rate of lumazine formation. Most notably, the rate of riboflavin formation has its maximum value at t = 0 and decreases dramatically after the consumption of PYR and DHB despite the presence of transiently formed lumazine. These data suggest that a significant fraction of DHB is converted to riboflavin by substrate channeling, which is conducive to an improved overall catalytic rate of riboflavin formation at low substrate concentrations. The channel is leaky, and the intermediate lumazine is therefore transiently accumulated in the bulk solution. The partitioning factor relating the direct formation of riboflavin via substrate channeling and the formation of transient 6,7-dimethyl-8-ribityl-lumazine increases at low concentrations of the substrates PYR and DHB and has a maximum value at pH 7.5. Channeling appears to result from the compartmentalization of the alpha subunits inside the icosahedral beta subunit capsid whose catalytic sites are located close to the inner capsid surface.

Biosynthesis of riboflavin. Studies on the reaction mechanism of 6,7-dimethyl-8-ribityllumazine synthase.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of 3 alpha subunits. The beta subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with (3S)-3,4-dihydroxy-2-butanone under formation of 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation yielding 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione as second product. (3R)- and (3S)-3,4-dihydroxy-2-butanone 4-phosphate were synthesized. Both enantiomers can serve as substrate for 6,7-dimethyl-8-ribityllumazine synthase. The reaction rate of the natural S-enantiomer is about 6-fold higher than that of the R-enantiomer. The Km value for (3S)-3,4-dihydroxy-2-butanone 4-phosphate is 130 microM, and the Km value for the pyrimidine substrate is 5 microM. Diacetyl and 3,4-dihydroxy-2-butanone 3-phosphate do not serve as substrates for lumazine synthase. The enzyme-catalyzed condensation of the carbohydrate with the pyrimidine is strictly regiospecific. The enzyme does not catalyze the exchange of protons between (3S)-3,4-dihydroxy-2-butanone 4-phosphate and solvent water in the absence of the pyrimidine cosubstrate. A reaction mechanism starting with the formation of a Schiff base followed by elimination of phosphate and cyclization is proposed. The lumazine synthase activities of the native enzyme complex and of reconstituted, hollow beta 60 capsids are virtually identical (about 12,000 nmol mg-1 h-1).

Conversion formulas between automated-perimetry indexes as measured by two different types of instrument.

PURPOSE: Comparison between the results of measurements made using different types of automated-perimetry equipment is difficult, because of the lack of standardisation. The purpose of the study was to compare the two main indexes, mean deviation or mean defect (MD) and pattern standard deviation (PSD), as measured by two widely used instruments (Interzeag Octopus 500EZ and Humphrey Field Analyser 740) in the same group of patients. PATIENTS AND METHODS: Thirty eyes of 17 patients with different stages of primary open-angle glaucoma were tested, and for 25 eyes the indexes measured using program G1 of the Octopus perimetry instrument were compared with those from program C30-2 of the Humphrey Field Analyser. RESULTS: Using the one-tailed Student t test, a significant difference was found between the MD and the PSD values of the Humphrey and of the Octopus perimetry equipments. However, subsequent use of the conversion formulas to adjust the measurements then indicated a good correlation between the two instruments (MD: r = 0.91; PSD: r = 0.79). CONCLUSION: MD and PSD values measured by the Octopus and Humphrey perimetry instruments are based on measurements made under significantly different conditions, so the direct comparison of the indexes is not reliable. Nonetheless, empirically valid conversion formulas exist and can be used to compare the perimetric results for a patient tested with the two types of instrument.

Biosynthesis of riboflavin. The reaction catalyzed by 6,7-dimethyl-8-ribityllumazine synthase can proceed without enzymatic catalysis under physiological conditions.

6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of the vitamin, riboflavin. The biosynthetic formation of the lumazine by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate is catalyzed by the enzyme, lumazine synthase. We show that the condensation reaction can proceed without enzyme catalysis in dilute aqueous solution at room temperature and neutral pH. The reaction rate is proportional to e (pH). The activation energy of the uncatalyzed reaction is E(a) = 46.3 kJ mol(-)(1). The regioselectivity of the uncatalyzed reaction increases with pH and temperature (70% at 65 degrees C and pH 7.75). The data suggest partitioning of the uncatalyzed reaction via two different reaction pathways. The value of k(cat)/k(uncat) may be indicative for an entropy driven process for the enzyme-catalyzed reaction.

Design and synthesis of 6-(6-D-ribitylamino-2,4-dihydroxypyrimidin-5-yl)-1-hexyl phosphonic acid, a potent inhibitor of lumazine synthase.

A novel inhibitor of lumazine synthase, the penultimate enzyme in the biosynthesis of riboflavin, has been synthesized. The inhibitor was designed by computer graphics molecular modeling using a hypothetical structure of the enzyme-inhibitor complex. The new compound is relatively potent when compared with the known inhibitors, and displays a KI of 109 microM.

Design, synthesis, and evaluation of 9-D-ribityl-1,3,7-trihydro-2,6,8-purinetrione, a potent inhibitor of riboflavin synthase and lumazine synthase.

Reduction of 5-nitro-6-D-ribitylaminouracil (9) afforded 5-amino-6-D-ribitylaminouracil (1), which reacted with ethyl chloroformate to yield 5-ethylcarbamoyl-6-D-ribitylaminouracil (12). The latter compound was cyclized to 9-D-ribityl-1,3,7-trihydropurine-2,6,8-trione (13), which was found to be a relatively potent inhibitor of both Escherichia coli riboflavin synthase (K(i) 0.61 microM) and Bacillus subtilis lumazine synthase (K(i) 46 microM). Molecular modeling of the lumazine synthase-inhibitor complex indicated the possibility for hydrogen bonding between the Lys135 epsilon-amino group of the enzyme and both the 8-keto group and the 4'-hydroxyl group of the ligand. A bisubstrate analogue of the riboflavin synthase-catalyzed reaction, 1,4-bis[1-(9-D-ribityl-1,3,7-trihydropurine-2,6,8-trionyl)]butane (18), was also synthesized using a similar route and was found to be inactive as an inhibitor of both riboflavin synthase and lumazine synthase.

Biosynthesis of vitamin b2 (riboflavin).

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. The imidazole ring of GTP is hydrolytically opened, yielding a 4, 5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3, 4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.

Biosynthesis of riboflavin.

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. Two reaction steps in the biosynthetic pathway catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase and riboflavin synthase are mechanistically very complex. The enzymes of the riboflavin pathway are potential targets for antibacterial agents.

Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein.

Recombinant Escherichia coli cells engineered for the expression of the xylB gene in conjunction with genes of the nonmevalonate pathway were supplied with (13)C-labeled 1-deoxy-D-xylulose. Cell extracts were analyzed directly by NMR spectroscopy. (13)C-labeled 2C-methyl-D-erythritol 2,4-cyclodiphosphate was detected at high levels in cells expressing xylB, ispC, ispD, ispE, and ispF. The additional expression of the gcpE gene afforded 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as an intermediate of the nonmevalonate pathway. Hypothetical mechanisms involving conserved cysteine residues are proposed for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalyzed by the GcpE protein.