Purification and characterization of a novel prolyl aminopeptidase from Maitake (Grifola frondosa)

We have found a novel prolyl aminopeptidase in Grifola frondosa. The enzyme was purified by DEAE-Sepharose CL-6B, Butyl-Toyopearl, Sephacryl S-100, and Mono-Q column chromatographies. The purified enzyme exists as a dimer and gives high activity toward L-proline-p-nitroanilide. The enzyme was strongly inhibited by p-chloromercuribenzoic acid and iodoacetic acid and markedly inhibited by phenylmethylsulfonyl fluoride and arphamenin A.     

Direct production of l-ornithine from casein by commercial digestive enzymes and in situ activated arginase

There is a great demand for l-ornithine, which is used as a dietary supplement, and in the pharmaceutical industry. In the present study, when milk casein was hydrolyzed at 37 °C by using commercial digestive enzymes, namely, Pancreatin F and Protease A, a significant accumulation of l-ornithine in the hydrolysate and the simultaneous disappearance of l-arginine was noted. In a radiometric assay, transient but distinct arginase activity, which was sufficiently high for l-ornithine production, was detected in the hydrolysate for a certain period during casein hydrolysis. On the basis of the results of the enzymatic analyses, arginase was thought to be proteolytically generated from an inactive precursor, which may generally be contained in Pancreatin F, and ultimately degraded by further proteolysis. This conversion process using the above-mentioned digestive enzymes is useful for the production of l-ornithine directly from protein sources that are abundant in nature.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Abstract Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae . It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Cytokine response and polymerase chain reaction study of peripheral blood mononuclear cells in infants with human cytomegalovirus infection

Abstract We tried to detect human cytomegalovirus (HCMV) DNA in CD4 + and CD8 + T lymphocytes from fourteen infants with HCMV hepatitis using polymerase chain reaction (PCR) assay. HCMV was isolated from their urine and anti-HCMV IgM antibody was detected in their sera. One set of primers were designed from a region — a major immediate early (IE) gene. We detected HCMV IE DNA in the specimens obtained from six infants. HCMV IE DNA was detected from CD4 + cells in two cases and from CD8 + cells in one. In three cases, HCMV IE DNA was detected from both CD4 + and CD8 + cells. We also studied the relationship between HCMV infection and serum levels of cytokines. We determined serum levels of interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α) and soluble interleukin 2 receptor (sIL-2R) which were associated with the activation of T lymphocytes by enzyme immunoassay. In the acute phase of HCMV infection, titers of sIL-2R were correlated with serum levels of liver enzymes in some cases. IL-4 and TNF-α activities were not detected in sera. It is likely that expression of viral genome on T lymphocytes as well as activities of some cytokines are associated with active HCMV infection.     

Detection of Human Cytomegalovirus DNA in Breast Milk by Means of Polymerase Chain Reaction

Three hundred and twenty-five breast milk samples were examined for the occurrence of human cytomegalovirus (HCMV) by cell culture method. Virus was isolated from the milk in 1 of 177 samples collected within 6 days after delivery, 2 of 115 samples collected during the period of 7 days to 1 month after delivery, 10 of 33 samples collected over 1 month after delivery. Next, we tried to amplify HCMV DNA from the breast milk samples from HCMV seropositive mothers and seronegative mothers at 1 month after delivery by polymerase chain reaction. HCMV DNA was detected in 12 of 13 samples from seropositive mothers and in none of 7 samples from seronegative mothers. It was thought that all women seropositive for HCMV principally shed the virus into their breast milk at 1 month after delivery.     

Enhanced phosphorylation and enzymatic activity of phosphoglucomutase by the Btk29A tyrosine kinase in Drosophila

The Drosophila Btk29A tyrosine kinase is suggested to be involved in diverse processes, although its target proteins are unknown. In the present study, we investigated substrates of Btk29A tyrosine kinase by expressing a catalytically activated form of Btk29A-P1 (Btk-EG) in Drosophila compound eyes. Expression in eye disks led to the development of the rough-eye phenotype and increased tyrosine phosphorylation of a 65-kDa protein. Partial amino acid sequence analysis of this protein showed that it was phosphoglucomutase. Phosphoglucomutase activity in heads from Btk-EG-expressing flies was higher than that in controls, suggesting that the levels of tyrosine phosphorylation and activity of the enzyme are associated with Btk29A tyrosine kinase activity.     

Immunocytochemical detection of enhanced expression of c-myc protein in the heart of cardiomyopathic hamster

An immunocytochemical study was performed to examine the expression of cellular c-myc protein in the heart of 30-, 120- and 180-day-old cardiomyopathic Syrian UM-X7.1 hamsters. The heart of age- and sex-matched BIO-RB hamster was used as normal control. In paraffin sections, an immunostaining for c-myc was markedly increased in cytoplasm of cells from the UM-X7.1 heart as compared with that of the BIO-RB heart which showed a weak staining. However, c-myc was localized in nuclei of cells in frozen sections of the heart. Specific cell types of the heart were differentiated with anti-vimentin, and we found that the increased expression of c-myc was present in nuclei of muscle cells of the UM-X7.1 myocardium. A quantitative study of c-myc-positive nuclei of muscle and nonmuscle cells was carried out by a video micrometer. The mean number of c-myc-positive nuclei of muscle cells was significantly higher in the cardiomyopathic heart than in the control heart from hamsters of all ages studied. These results suggest that the increase of c-myc protein may relate to the pathological state or pathogenesis of the hereditary cardiomyopathy.