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BACKGROUND: Psoriatic arthritis concerns both the skin and the joints. Therapeutic interventions should therefore ideally improve both symptoms. Current disease modifying drugs are effective; however, they are usually limited to either psoriasis or arthritis. AIM OF THE STUDY: The aim of this study was to analyze the therapeutic effects of a new immunosuppressive drug (Mycophenolate mofetil) in the treatment of psoriatic arthritis. METHODS: Six patients with psoriatic arthritis were treated with Mycophenolate mofetil for a period of 12 weeks and examined every 14 days (range of motion, joint swelling, joint deformity, PASI score (Psoriasis Area and Severity Index). In addition, a life quality assessment (SF-36 Health Survey) was performed. White and red blood count as well as inflammation parameter were controlled regularly. RESULTS: 6 patients could be included in a complete follow-up (5 men, 1 woman, average age 50.3 years, average duration of psoriasis 8.3 years, average duration of arthritis 5.7 years). Four of 6 patients showed relevant improvement in pain, mobility and degree of psoriatic skin effluorescences. Only 3 of 6 showed relevant improvement in life quality. CONCLUSION: For the first time results of treatment of psoriatic arthritis with a new immunosuppressive drug (Mycophenolate mofetil) were presented. A positive influence on both, the arthritis and the psoriasis could be shown. These first observations warrant controlled, randomized clinical trials.

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The aim of this study was to define prognostic factors for survival, and especially for long-term survival in a mature data-set of patients with stage IV melanoma treated within a randomised trial of cytokine-based protocols. Long-term follow-up data on patients enrolled into a European Organization for Research and Treatment of Cancer (EORTC) trial comparing interferon-alpha (IFNalpha) plus interleukin-2 (IL-2) with or without cisplatin were collected. Univariate and multivariate Cox regression analyses were performed to define prognostic factors for survival. The characteristics of patients alive at 2 and 5 years after randomisation were compared with the entire cohort using the chi(2) test. The minimum potential follow-up of the 131 evaluable patients was 5 years. 18 patients (14%) were alive 2 years after randomisation, and 11 (8%) 5 years after randomisation. Pretreatment performance status (PS), serum lactate dehydrogenase (LDH) and tumour mass were significant predictors for survival, whereas site of metastases and number of sites were non-significant. PS and LDH were the only independent prognostic factors. All except 1 patient alive at 2 and 5 years had a pretreatment PS of 100%, and only three long-term survivors had elevated pretreatment LDH. There was no association between the site of metastases and long-term survival. Response to treatment was a major predictor for long-term survival, whereas addition of cisplatin did not impact upon overall survival probability or on long-term survival. The probability of long-term survival in stage IV melanoma patients after IL-2-based treatments is governed by pretreatment PS, serum LDH and response to treatment. Site of metastases, the basis for the M-subcategories of the new AJCC staging system, was not informative in this study.

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A 63 year old man presented with circumscribed myxedema surprisingly involving both the arms and legs. He had a history of thyroiditis with hyperthyroidism and an infiltrative ophthalmopathy leading to the diagnosis of Graves disease. At the time of presentation the patient had already been treated with radioactive iodine and undergone endonasal orbital decompression. We review the pathogenesis and treatment of Graves disease and discuss the first therapeutic attempt with PUVA-bath therapy.

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Dental caries has been an intractable disease in spite of intense dental research. The metabolic acids produced by mutans streptococci demineralize the tooth surface and lead to dental caries. The enzyme glucosyltransferase (GTF) produced by mutans streptococci is the key factor in this process. Oral bacterial GTFs use sucrose as a substrate in synthesis of either water-soluble or insoluble glucans. In this investigation, kinetic studies with divalent metal ions revealed their strong binding affinity to GTF. The metal ions also proved to be strong inhibitors of the enzyme. Here we describe a simple method of inactivating the enzyme that actively participates in dental caries by taking advantage of a Fenton reaction which requires metal ions such as iron or copper and peroxide. The hydroxyl radical ions produced via the Fenton reaction inactivate GTF, a factor in the production of dental caries.

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Glucosyltransferase from oral bacteria Streptococcus mutans is the most significant virulent factor in causing dental caries. The enzyme has two subsites. The binding specificity of divalent metal ions to glucosyl or fructosyl subsite was examined using multiple inhibition kinetics. The interaction factor "alpha" identifies whether the two subsites are exclusive or non-exclusive.

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Mycophenolate mofetil (MMF), a widely used immunosuppressant in organ transplantation, is a recent addition to the therapeutic armamentarium of autoimmune and inflammatory skin disorders in dermatology. We describe 5 patients with moderate to severe chronic plaque psoriasis and 6 patients with psoriatic arthritis that was refractory to conventional systemic and/or topical antipsoriatic treatment who were treated with MMF monotherapy (2 g/d) in a 10-week study. Although MMF was tolerated well in all patients, only patients with moderate psoriasis and psoriatic arthritis improved with therapy, whereas patients with severe psoriasis did not respond to MMF. Although MMF seems to be effective and safe for blistering autoimmune diseases and pyoderma gangrenosum, our data do not allow optimistic statements on the use of MMF in severe plaque-stage psoriasis. However, MMF may develop into an interesting therapeutic alternative for patients with psoriatic arthritis.

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HISTORY AND CLINICAL FINDINGS: A 39-year-old man was admitted for treatment of bilateral inflammatory-pustular skin changes in the area of the large toes and soles of the feet. Antibiotic treatment and an Emmert wedge resection had already been unsuccessfully performed at another hospital for what was diagnosed as paronychia. On admission there were inflammatory, in part erosive, red areas with yellow and partly confluent pustules on the distal phalanges of both great toes. The entire right nail-bed and left medial nail-bed were missing. In the area of the capillitium, both lower arms and the sulcus coronarius there were erythematous squamous plaques. INVESTIGATIONS: Radiography of the great toes demonstrated dystrophic demineralisation, in part with subchondral cystic changes of the spongiosa. Histological examination of the nail-bed showed hyperplasia and papillomatosis, definite hyperkeratosis with a prominent granular layer, as well as ortho- and parahyperkeratosis. Laboratory tests for inflammatory disease were unremarkable and there was no association with HLA B27. DIAGNOSIS, TREATMENT AND COURSE: Suppurative acrodermatitis continua of Hallopeau was diagnosed and immunosuppressive treatment with cyclosporin A given (initially 4.4 mg/kg. stepwise reduction to 2.5 mg/kg within 6 weeks, this dosage then continued for a further 10 weeks). Nearly complete healing was achieved, but the condition recurred in a mild form 2 weeks after the end of treatment. CONCLUSION: Suppurative acrodermatitis continua of Hallopeau should be included in the differential diagnosis of inflammatory changes of the distal phalanges.

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Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of alpha-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with alpha-amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/alpha-amylase residues conserved in all but one alpha-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the alpha-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (beta/alpha)8-barrel structure and belong to family 13 of the glycohydralases.

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Oral bacterial glucosyltransferases use sucrose as a substrate in synthesis of either alpha-1,3 water-insoluble glucans (GTF-I) or alpha-1,6 water-soluble glucans (GTF-S). The binding specificity of the glucosyl and fructosyl subsites of the sucrose-binding site was examined to identify ligands that bind exclusively to each subsite. Such compounds can be used as reporter ligands to localize the subsite binding of any reversible or irreversible active site inhibitor. In examining potential subsite-specific ligands, binding affinity to GTF-I was consistently stronger than binding to GTF-S. Fructose was found to be a moderate GTF inhibitor, but free glucose, alpha-methylglucoside and glucose epimers were very weak inhibitors. In contrast, glucose transition-state analogues, D-glucano-1,5-lactone, 1-deoxynojirimycin (dNJ), and most N-alkyl derivatives of dNJ were moderate to strong inhibitors; in particular N-methyl-dNJ was found to be the strongest GTF inhibitor identified to date. Multiple inhibitor kinetic analysis established nonexclusive binding of fructose and dNJ at the respective subsites. Binding of fructose and N-alkyl-dNJ derivatives was, to a small degree, partially exclusive. Fructose and dNJ were used as reporter ligands to localize the subsite specificity of two test inhibitors: a reversible inhibitor, Zn2+, and an irreversible inhibitor, diethyl pyrocarbonate (DEP). Zn2+ paired with dNJ in multiple inhibitor kinetic analysis showed no competition between the inhibitors, while Zn2+ paired with fructose decreased ligand affinity 7-fold, establishing Zn2+ binding exclusively at the fructose subsite. Analogous experiments adapted to the irreversible inhibitor DEP indicated that it reacts at both subsites or induces a protein conformational change at one subsite that alters ligand binding at the adjacent subsite.

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Polyclonal antibodies were raised against peptides derived from an active-site sequence common to the family of mutans streptococcal glucosyltransferases (GTFs). The sequence contains an aspartic acid residue that functions in formation of the enzyme transition state in catalysis. Two GTFs were targeted with similar but not identical sequences in this region: one that synthesizes an alpha-1,3-linked water-insoluble glucan and a homologous GTF that synthesizes an alpha-1,6-linked water-soluble glucan. For each enzyme, an 8-mer and 22-mer peptide were prepared. The two peptide lengths were chosen in order to increase the likelihood of the peptides folding in a conformation similar to that of the native enzyme. Each peptide immunogen produced high titers of antibody in rabbits, and all antisera cross-reacted with all peptides, albeit to various degrees. Native enzyme showed weak interaction with antisera, which, on the basis of enzyme denaturation experiments, likely reflects binding to a small but finite population of denatured enzyme in the sample. GTF was assayed for inhibition in the presence of protein A-purified immunoglobulin G from each antiserum. Given the mass of the antibody and catalytic importance of the peptide, any enzyme-antibody complex formation would result in enzyme inhibition. No significant inhibition was observed, which demonstrates that either polyclonal antibodies raised against each of the four peptides cannot access this active-site region, or antibodies do not recognize the native enzyme conformation. The advantages and challenges of generating antibodies against enzyme active-site peptides are discussed in the context of the crystal structure of Aspergillus oryzae alpha-amylase, which has a homologous peptide segment which serves the same catalytic function.

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An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases.

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Mild trypsin proteolysis of Streptococcus sobrinus sucrose:3-alpha-D-glucosyltransferase (GTF-I) reduced most of the enzyme to small products but left a few large fragments undigested. The digest had no glucosyl transfer activity, but several digestion products had an affinity for glucan equivalent to that of the native enzyme. The glucan-binding fragments ranged in size from 17 to 60 kilodaltons (kDa), with a particularly prominent 42-kDa fragment. The largest of these (60 kDa) appears to be the full extent of the domain since it increases in abundance when the enzyme is protected with glucan during proteolysis. The presence of smaller fragments with glucan-binding function and intact tertiary structure indicates that the full domain must be built on glucan-binding subdomains. Among the range of glucan-binding fragments, only the 42-kDa segment could be satisfactorily purified. It was subjected to N-terminal sequence analysis and, because of some ambiguity, was also subjected to chymotrypsin digestion and sequence of several chymotryptic peptides. The sequence data established that the 42-kDa domain fragment is initiated approximately two-thirds into the 170-kDa enzyme in a region previously identified as a segment of the gene that includes the glucan-binding domain (J. J. Ferretti, M. L. Gilpin, and R. R. B. Russell, J. Bacteriol. 169:4271-4278, 1987). The site is approximately 60 kDa from the C terminus and covers a region characterized by extensive amino acid sequence repeats. The data are discussed in the context of the size range of the glucan-binding fragments and subdomain architecture of the full glucan-binding domain.

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A covalent glucosyl-enzyme was isolated from a quenched reaction of Streptococcus sobrinus sucrose 6-alpha-D-glucosyltransferase and radiolabeled sucrose. No complex was observed with heat-inactivated enzyme or when sucrose was replaced with radiolabeled maltose or glucose. The complex was stable at pH 2 in 1% sodium dodecyl sulfate, 6.0 M urea, and 4.0 M guanidine hydrochloride, but became increasingly labile with increased pH (32-min half-life at pH 7.0). D-Glucose was the exclusive radiolabeled compound identified when all radioactivity was released under mild alkaline conditions. Glucosyl-enzyme hydrolysis rates were linearly dependent on hydroxide ion concentration, giving a second-order rate constant of 2.15 x 10(5) M-1 min-1. When compared to the base lability of known glycosyl amino acid derivatives, the pH dependency of the glucosyl-enzyme most closely paralleled a glucosyl linkage to a carboxyl group. A novel application of a carbohydrate high-performance liquid chromatography column in aqueous solution was used to identify the anomeric form of D-glucose released on (i) alkaline hydrolysis of denatured glucosyl-enzyme and (ii) native enzyme hydrolysis of sucrose. The beta-anomer was identified in the former case and the alpha-anomer in the latter. The results with the denatured glucosyl-enzyme are consistent with a beta-glucosyl ester linkage to an aspartic or glutamic acid that hydrolyzes at the ester carbon with retention of anomeric configuration; for native glucosyltransferase catalysis, the data are consistent with a beta-glucosyl covalent intermediate as well, where deglucosylation occurs by attack at the acetal carbon with anomeric inversion.(ABSTRACT TRUNCATED AT 250 WORDS)

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A glucan-binding domain of 1,6-alpha-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.

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A direct method for the determination of N-linked glycosylation sites in highly glycosylated proteins is described. Carcinoembryonic antigen (CEA) and a nonspecific crossreacting antigen (NCA) were chemically deglycosylated, and peptide maps were prepared by reverse-phase HPLC. The peptides were sequenced on a gas-phase microsequencer, and glycosylation sites were identified as the phenylthiohydantoin derivative of N-acetylglucosaminylasparagine. The sequences were confirmed by fast atom bombardment mass spectrometry. Highly homologous, extended amino-terminal sequences were determined for CEA and two NCAs, NCA-95 and NCA-55. Cysteine-containing sequences for CEA and NCA-95 show up to 95% sequence homology, and the CEA sequences also show internal sequence homologies. A comparison of the CEA sequences with known protein sequences suggests that CEA may be a member of the immunoglobulin supergene family. The protein sequence data have been used to identify a genomic DNA clone for one of the NCA antigens [Thompson, J., Pande, H., Paxton, R. J., Shively, L., Padma, A., Simmer, R. L., Todd, C. W., Riggs, A. D. & Shively, J. E. (1987) Proc. Natl. Acad. Sci. USA, in press] and a cDNA clone for CEA [Zimmermann, W., Ortlieb, B., Friedrich, R. & von Kleist, S. (1987) Proc. Natl. Acad. Sci. USA, in press].

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The kinetic mechanism of dextransucrase was studied using the Streptococcus mutans enzyme purified by affinity chromatography to a specific activity of 36.9 mumol/min/mg of enzyme. In addition to dextran synthesis, the enzyme catalyzed sucrose hydrolysis and isotope exchange between fructose and sucrose. The rates of sucrose hydrolysis and dextran synthesis were partitioned as a function of dextran concentration such that exclusive sucrose hydrolysis was observed in the absence of dextran and exclusive dextran synthesis at high dextran concentrations. An analogous situation was observed with fructose-dependent partitioning of sucrose hydrolysis and fructose exchange. Steady state dextran synthesis and fructose isotope exchange kinetics were simplified by assay at dextran or fructose concentrations high enough to eliminate significant contributions from sucrose hydrolysis. This limited dextran synthesis assays to dextran concentrations above apparent saturation. The limitation was diminished by establishing conditions in which the enzyme does not distinguish between dextran as a substrate and product which allowed initial discrimination among mechanisms on the basis of the presence or absence of dextran substrate inhibition. No inhibition was observed, which excluded ping-pong and all but three common sequential mechanisms. Patterns of initial velocity fructose production inhibition and fructose isotope exchange at equilibrium were consistent with dextran synthesis proceeding by a rapid equilibrium random mechanism. A nonsequential segment was apparent in the exchange reaction between fructose and sucrose assayed in the absence of dextran. However, the absence of detectable glucosyl exchange between dextrans and the lack of steady state dextran substrate inhibition indicate that glucosyl transfer to dextran must occur almost exclusively through the sequential route. A review of the kinetic constants from steady state dextran synthesis, fructose product inhibition, and fructose isotope exchange showed a consistency in constants derived from each reaction and revealed that dextran binding increases the affinity of sucrose and fructose for dextransucrase.

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