Identification of the peptide motifs that interact with HLA-DR8 (DRB1*0802) in Streptococcus mutans proteins.

A glucosyltransferase (GTF) and a surface protein antigen (PAc) of Streptococcus mutans have been suggested as possible components of an effective dental caries vaccine. To identify antigenic peptides in GTF and PAc that bind to MHC class II (HLA-DR8, DRB1*0802) molecules, we investigated binding activities to DR8 molecules of overlapping synthetic peptides at several sites in GTF and in the alanine-rich repeating region of PAc using an ELISA-inhibition competitive binding assay for the interaction between the HLA-DR molecule and the PAc (316-334) peptide. Six GTF peptides and 10 PAc peptides strongly bound to the HLA-DR8 molecule. In a homology analysis of the amino acid sequences of the six GTF peptides, two binding motifs were found in L/Y--Y/L-A/N and Y/L--N/G/E--Y-V/L/P. Moreover, a new binding motif in PAc was found in L--Y-A. It is suggested that these binding motifs could be useful in designing a dental caries vaccine in humans.

Pericardial cyst in a child treated with video-assisted thoracoscopic surgery.

UNLABELLED: A pericardial cyst is a rare condition in childhood. We report on a 10-year-old girl who presented with an intrathoracic mass detected on a chest X-ray performed during a routine medical examination. She had no symptoms and a physical examination revealed no abnormalities. Ultrasonography, computed tomography and magnetic resonance imaging showed a multiloculated cystic mass in the right upper thorax. The cyst was resected using a thoracoscopic procedure. Histologically, the findings were consistent with a pericardial cyst. Thoracoscopic surgery was an effective surgical technique even for such a young patient and the results successfully reduced the morbidity. CONCLUSION: A pericardial cyst, a rare condition in childhood, was treated successfully by video-assisted thoracoscopic surgery.

Inhibitory effect of a self-derived peptide on glucosyltransferase of Streptococcus mutans. Possible novel anticaries measures.

Glucosyltransferase (GTF) plays an important role in the development of dental caries. We examined the possible presence of self-inhibitory segments within the enzyme molecule for the purpose of developing anticaries measures through GTF inhibition. Twenty-two synthetic peptides derived from various regions presumably responsible for insoluble-glucan synthesis were studied with respect to their effects on catalytic activity. One of them, which is identical in amino acid sequence to residues 1176-1194, significantly and specifically inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Double-reciprocal analysis revealed that the inhibition is noncompetitive. Scramble peptides, composed of the identical amino acids in randomized sequence, had no effect on GTF-I activity. Furthermore, the peptide is tightly bound to the enzyme once complexed, even in the presence of sodium dodecyl sulfate (SDS). Kinetic analysis using an optical evanescent resonant mirror cuvette system demonstrated that the enzyme-peptide interaction was biphasic. These results indicate that the peptide directly interacts with the enzyme with high affinity and inhibits its activity in a sequence-specific manner. This peptide itself could possibly be an effective agent for prevention of dental caries, although its effectiveness may be improved by further modification.

A defect in GTP synthesis affects mannose outer chain elongation in Saccharomyces cerevisiae.

We have found that yeast mutants that are defective in mannose outer chain elongation of N-linked glycoproteins show higher cell wall porosity than normal cells, and are hypersensitive to antibiotics with a large molecular weight; such as neomycin and geneticin. Wild-type yeast cells also showed enhanced sensitivity to neomycin in the presence of tunicamycin, an inhibitor of N-glycosylation, suggesting that the extent of N-glycosylation may affect the sensitivity of yeast cells to drugs and that sensitivity to neomycin may be an effective method for screening for yeast mutants defective in N-glycosylation. Pursuing this logic, we isolated neomycin-sensitive yeast mutants and screened them for defects in N-glycosylation. The neomycin-sensitive, N-glycosylation-defective mutants fell into 15 complementation groups including alleles of the previously isolated temperature-sensitive nes mutants nes10, nes17, and nes25. Gene cloning revealed that NES10 was identical to SEC20, which is involved in ER-Golgi protein transport, NES17 was identical to ALG1, which encodes a beta-1,4-mannosyltransferase present in the ER, MSN17, a multicopy suppressor of nes17/alg1, was also isolated and found to be an allele of PSA1, which is involved in GDP-mannose synthesis, NES25 was identical to GUK1, which encodes a GMP kinase. Overexpression of MSN17 increased the GDP-mannose level in a wild-type strain by about threefold, and guk1 decreased the GDP-mannose level to one-fourth, suggesting a close relationship between GTP metabolism and mannose outer chain elongation; the link is presumably provided by the process of GDP-mannose transport in the Golgi membranes.

Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 3: Evaluation as candidate working enzyme reference material for gamma-glutamyltransferase.

Seraclear-HE, containing gamma-glutamyltransferase (GGT, EC 2.3.2.2) derived from the cell culture of a human macrophage cell line, was evaluated as a candidate enzyme reference material (ERM) to calibrate routine methods in terms of a Reference Method for GGT. We have compared this preparation with commercially available materials, including Certified Reference Material 319, at 30 degrees C and 37 degrees C with respect to kinetic properties and with respect to commutability between the Reference Method and each of 15 analytical procedures involving five structurally different donor substrates. GGTs of human origin are far more commutable with reagents of varied types than are GGTs derived from animals. Calibration of 44 patients' sera with Seraclear-HE decreased average intermethod variation from 20% to approximately 4%, whereas GGTs of animal origin showed intermethod variations of approximately 30% with no benefit from calibration. Seraclear-HE is promising as a secondary or working ERM to be used as an intermethod calibrator.

The role of the calpain-calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, nPKC epsilon, in rat pituitary GH4C1 cells.

We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals.

Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 2: Evaluation as candidate working enzyme Reference Material for alanine and aspartate aminotransferases.

Seraclear-HE, containing seven enzyme analytes from human sources, was evaluated as an intermethod calibrator for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to transfer Reference Method values to seven routine methods, including one based on hydrogen peroxide detection for possible unification of values (interlaboratory comparability of data). The commutabilities of AST from erythrocytes and ALT from a hepatoma cell line were studied between the consensus methods of Japan Society of Clinical Chemistry (chosen as the Reference Methods) and each of the automated routine methods at reaction temperatures of 30 degrees C and 37 degrees C. For AST, calibration of patients' sera with Seraclear-HE decreased average intermethod variation (CV) from 12% to 2%; for ALT, the decrease was from 20% to 3%. For both enzymes, Seraclear-HE was judged to be commutable between the Reference Methods and each of the methods investigated. The limitations for such use are discussed.

Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 1: Preparation and properties.

We have developed a new multienzyme control serum, Seraclear-HE, which was designed to function not only as an accuracy and precision control serum but also as an intermethod calibrator for unifying interlaboratory clinical enzyme data in terms of reference method values. Seraclear-HE contains as analytes the following enzymes of human origin only: aspartate aminotransferase (AST, EC 2.6.1.1) and lactate dehydrogenase (LD, EC 1.1.1.27) from erythrocytes; alanine aminotransferase (ALT, EC 2.6.1.2) from a hepatoma cell line; alkaline phosphatase (ALP, EC 3.1.3.1) from an amnion cell line; creatine kinase (CK, EC 2.7.3.2) from an embryo kidney cell line; gamma-glutamyltransferase (GGT, EC 2.3.2.2) from a macrophage cell line; and amylase (AMY, EC 3.2.1.1) from urine and saliva. The seven partly purified enzymes were lyophilized in partially delipidated human serum containing sucrose (50 g/L), pyridoxal 5'-phosphate (30 mmol/L), and other stabilizers. The material is stable for at least 2 years at temperatures < or = 10 degrees C. For two concentrations of this preparation, reference method values (mainly International Federation of Clinical Chemistry and Japan Society of Clinical Chemistry) obtained at both 30 degrees C and 37 degrees C are assigned.

Multi-enzyme reference material from established human cell lines and human sources.

A multi-enzyme reference material was prepared from seven enzymes of asparatate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), alkaline phosphatase (ALP, EC 3.1.3.1), lactate dehydrogenase (LD, EC 1.1.1.27), creatine kinase (CK, EC 2.7.2.2), gamma-glutamyltranspeptidase (gamma-GT, EC 2.3.2.2) and amylase (AMY, EC 3.2.1.1) which were purified from human sources including established human cell lines. The enzymatic properties of the material closely resembled those of human serum. In lyophilized form the preparation was stable for at least 200 days when stored at 40 degrees C. Intermethod comparisons of the enzyme activities in 80 clinical specimens were done by correcting the mean values with calibration constants for different assay methods resulting from use of a human serum, the multi-enzyme reference and a commercial control serum. The results from the comparison for the six enzymes of AST, ALT, LD, CK, gamma-GT and AMY in use of the multi-enzyme reference were almost the same as those with use of a human serum as a calibrator, but were not satisfactory for ALP. Even though further search for more reliable material for ALP is required the multi-enzyme reference material can be used for standardization in clinical chemistry.

[Measurement of enzyme activity of control materials containing human enzymes by IFCC reference methods].

In order to unify interlaboratory clinical enzyme data, a reference method and reference material are necessary for each enzyme. Although reference methods recommended by various societies of clinical chemistry including JSCC are now available for some routinely measured clinical enzymes, the availability of secondary enzyme references or calibrators, limits the practical application of standardization concept for minimizing present interlaboratory variation of enzyme data. Since control sera prepared by adding enzymes from human cell cultures to a pool of human serum may meet the requirements as candidates for such reference materials, we attempted to assign values to 6 enzymes present in the materials by mainly the IFCC reference methods following the procedures as strictly as possible and yet with modifications where necessary. This paper describes each step of the value assignment for accuracy control purposes.