Angiotensin II-producing proteases from granulomatous tissue reaction in mice infected with Schistosoma mansoni

1. Angiotensin I hydrolases, Mr 140,000 and Mr 70,000 were separated by gel filtration from Tris-HCl buffer extract of hepatic granulomas developed in mice with schistosomiasis. Two enzymes had different substrate specificity. 2. Mr 140,000 hydrolase activity was inhibited by captopril as reported for angiotensin converting enzyme (ACE), while that of Mr 70,000 hydrolase activity was inhibited by potato carboxypeptidase inhibitor. 3. An intermediary, des-Leu10-angiotensin I and then angiotensin II were formed from angiotensin I by Mr 70,000 hydrolase. 4. The findings suggest that Mr 70,000 enzyme is tissue carboxypeptidase A, and it generates angiotensin II in granulomatous inflammation as does ACE.     

Regional localization of the hepatocyte growth factor (HGF) gene to human chromosome 7 band q21.1.

Hepatocyte growth factor (HGF) is a polypeptide involved in liver regeneration. Its amino acid sequence and gene structure are similar to those of coagulation-related serine proteases. We have used a cDNA clone of HGF and flow-sorted human chromosomes to assign this gene to chromosome 7. Fluorescence in situ hybridization of the HGF genomic clones to human metaphase chromosome spreads showed the localization of this gene to 7q21. Estimation of fluorescent signals relative to arbitrary reference points (ARPs) allowed further localization to 7q21.1.     

An oxygraphic method for the determination of cholesterol and measurement of the slow oxygen-consuming reactions of hepatic microsomes.

Oxygen-consuming reactions of cholesterol oxidase [EC 1.1.3.6] and microsomes were measured with a galvanic oxygen electrode which was attached to an offset amplifier for sensitive measurement of the reaction processes. The sensitivity of this oxygraphic method for detection of oxygen consumption was ten times greater than that of the usual method. The minimum rate of slow oxygen-consuming reactions which could be estimated was about 5 nmoles of oxygen per min, and the minimum amount of oxygen consumption which could be determined was also about 5 nmoles. An oxygraphic method for direct and rapid determination of cholesterol was demonstrated using one-twentieth the amount of cholesterol oxidase which is used for the colorimetric method. The processes of cyanide-suppressed beta-NADH-dependent oxygen consumption and cyanide-insensitive alpha-NADH-dependent oxygen consumption, which were difficult to follow by the usual method, were followed using a small amount of microsomes (less than 1mg protein/ml). Furthermore, the temporary cessation of alpha-NADH-dependent oxygen consumption caused by ferricyanide and the corresponding oxidation-reduction of reduced cytochrome b5 were followed in the presence of ADP. ADP did not inhibit the oxygen consumption. The results indicate that the oxygen consumption with alpha-NADH is due to electron transfer from alpha-NADH via NADH-cytochrome b5 reductase and cytochrome b5, in which the rate-determining step lies at some reaction after the reduction of cytochrome b5.     

Immunofluorescent studies of epidermal protein during UV induced carcinogenesis.

Previous studies with agar diffusion technique demonstrated that antibodies produced in rabbits by injection of urea extractable proteins of rat cornfied cells cross react with proteins extracted from normal epidermis of hairless mice using the same technique. In the present study we investigated by indirect immunofluorescence microscopy the immunoreactivity of epidermal proteins in normal and ultraviolet light (UVB) induced hyperplasia and malignant transformation. Reactivity to the antibody was seen over the entire epidermis of nontreated skin and hypertrophied epidermis which occurred at 6-8 weeks after initiation of UVB irradiation. However, the reactivity diminished when malignant changes took place in the epidermal cells. Almost complete disappearance of the immunoresponse was observed in squamous cell carcinoma produced by further UVB radiation. These results suggest that the reactivity of this urea extractable protein serves as an additional immunologic marker for normal epidermal cells. Alterations in the immunoreactivity parallels UVB induced carcinogenesis.     

Immunological detection of a cysteine protease in the skin and other tissues

Summary Monospecific antibody directed to cysteine protease of 2-day-old rat epidermis recently characterized as being different from the proteases previously reported was produced in rabbits. By immunofluorescence microscopy and immunoperoxidase staining with an avidin-biotin-peroxidase method the protease was found to be present in the epidermis of rodents of different ages as well as that of humans, but not in the dermis. The staining in germinative cells was more intense than in cells in the superficial layers. It appeared as irregular patches in the nuclei and stained more diffusely in the cytoplasm where small granular components, strongly stained, were identified. The staining patterns in granular cells showed accumulation of the antigen in a granular form. The morphology and distribution of granules resembled those of keratohyalin-like granules in the nucleus and dense homogenous deposits in the cytoplasm. In cornified cells the reaction product was localized by the plasma membrane where concentration of the dense homogenous deposits occurred, suggesting that the cysteine protease is one component of the unique and characteristic structure of differentiated keratinocytes. In addition, the cysteine protease antigen having the same molecular weight as the epidermal enzyme was detected in liver, kidney and lung indicating a wider tissue distribution of the protease. The significance of the protease in regulation of cellular functions remains to be investigated.     

Adult onset globoid cell leukodystrophy (Krabbe disease): analysis of galactosylceramidase cDNA from four Japanese patients

We examined galactosylceramidase (GALC) cDNA in four Japanese patients with adult onset globoid cell leukodystrophy (Krabbe disease; AO-GLD) by polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis, subsequent sequence determination, and restriction enzyme digestion of PCR products. Initial symptoms were the onset of slowly progressive spastic paraplegia from the middle of the second decade, and all patients had diminished GALC activity in their leukocytes. We identified three missense mutations (I66M, G270D, L618S) and one exon-6 skipping (535– 573del). Two of the patients had only the I66M mutant mRNA, and one only the G270D mutant mRNA. The fourth patient carried a compound heterozygous mutation of 535–573del and L618S. To determine the enzymatic activities produced by these mutations, we constructed mutated GALC cDNAs and expressed them in COS-1 cells. Three mutations, viz., G270D, L618S, and exon-6 skipping (535–573del), produced diminished GALC activity as expected. The I66M mutation in the wild-type GALC cDNA(I289) had normal activity, but when this mutation and the V289 polymorphism were introduced into the same allele, it had decreased activity. Thus, the combination of a unique mutation and polymorphism causes conformational change in the GALC enzyme, resulting in low enzymatic activity. AO-GLD mutations, including those found here, are located in the N-terminus (I66M, G270D, 535–573del) or C-terminus (L618S) of the GALC enzyme, whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the clinical features of GLD. Received: 4 February 1997 / Accepted: 28 April 1997     

Genetic Variations in the Human Cannabinoid Receptor Gene Are Associated with Happiness

Happiness has been viewed as a temporary emotional state (e.g., pleasure) and a relatively stable state of being happy (subjective happiness level). As previous studies demonstrated that individuals with high subjective happiness level rated their current affective states more positively when they experience positive events, these two aspects of happiness are interrelated. According to a recent neuroimaging study, the cytosine to thymine single-nucleotide polymorphism of the human cannabinoid receptor 1 gene is associated with sensitivity to positive emotional stimuli. Thus, we hypothesized that our genetic traits, such as the human cannabinoid receptor 1 genotypes, are closely related to the two aspects of happiness. In Experiment 1, 198 healthy volunteers were used to compare the subjective happiness level between cytosine allele carriers and thymine-thymine carriers of the human cannabinoid receptor 1 gene. In Experiment 2, we used positron emission tomography with 20 healthy participants to compare the brain responses to positive emotional stimuli of cytosine allele carriers to that of thymine-thymine carriers. Compared to thymine-thymine carriers, cytosine allele carriers have a higher subjective happiness level. Regression analysis indicated that the cytosine allele is significantly associated with subjective happiness level. The positive mood after watching a positive film was significantly higher for the cytosine allele carriers compared to the thymine-thymine carriers. Positive emotion-related brain region such as the medial prefrontal cortex was significantly activated when the cytosine allele carriers watched the positive film compared to the thymine-thymine carriers. Thus, the human cannabinoid receptor 1 genotypes are closely related to two aspects of happiness. Compared to thymine-thymine carriers, the cytosine allele carriers of the human cannabinoid receptor 1 gene, who are sensitive to positive emotional stimuli, exhibited greater magnitude positive emotions when they experienced positive events and had a higher subjective happiness level.     

Novel NGF-potentiating diterpenoids from a Brazilian medicinal plant,Ptychopetalum olacoides

From the MeOH extract of Ptychopetalum olacoides, which is used in Brazilian folk medicine for the treatment of chronic degenerative conditions of the nervous system, four novel clerodane-type diterpenoids named 6α,7α-dihydroxyannonene (1), 7α,20-dihydroxyannonene (2), 7α-hydroxysolidagolactone I (3), and ptycho-6α,7α-diol (4) were isolated by bioassay-directed fractionation using NGF-differentiated PC12 cells. The structures of 1–4 were established by extensive NMR spectroscopic analyses and chemical conversion. Compounds 1 and 2 significantly enhanced NGF-mediated neurite outgrowth in PC12 cells at concentrations ranging from 0.1 to 50.0 μM for 1 and 0.1 to 30.0 μM for 2, whereas 3 and 4 had no morphological effect on NGF-mediated PC12 cells in the same concentration range. The structure–activity relationship of these compounds is also discussed.     

Structure of the [2Fe-2S] ferredoxin I from the blue-green alga Aphanothece sacrum at 2.2 A resolution

Crystals of a [2Fe-2S] ferredoxin (Fd) I with a relative molecular mass of 10,480 were obtained from the blue-green alga Aphanothece sacrum. Each asymmetric unit of the crystal contains four molecules. An electron density map calculated by the single isomorphous replacement method with the anomalous dispersion at 2.5 A resolution was refined by averaging the four molecules in the asymmetric unit. Positional and isotropic thermal parameters for the non-hydrogen atoms of the four molecules and 158 water molecules were refined to an R-factor (R = sigma[Fo-Fc[/sigma Fo) of 0.23 by the restrained least-squares method. The estimated root-mean-square (r.m.s.) error for the atomic positions is 0.3 A. The r.m.s. deviations of equivalent C alpha atoms of the asymmetric-unit molecules superposed by the least-squares method average 0.35 A. The Fd molecule has a structure like the beta-barrel in the molecule of the [2Fe-2S] Fd from Spirulina platensis. A [2Fe-2S] cluster is bonded covalently to the protein molecule by four Fe-S, in which three of the Fe-S bonds are in a loop segment from position 38 to 47. The hydrophobic core inside the beta-barrel is formed by seven conservative residues: Val15, Val18, Ile24, Leu51, Ile74, Ala79 and Ile87. The molecular surface around Tyr23, Tyr80 and the active center may interact with ferredoxin-NADP+ reductase. One of the two iron atoms of the [2Fe-2S] cluster should be more easily reduced than the other because of differences in the hydrogen-bonding scheme and the hydrophobicity around the atoms.