Penicillopepsin, the aspartic proteinase from Penicillium janthinellum: substrate-binding effects and intermediates in transpeptidation reactions.
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Biomedical subjects
Publications and source records attributed to A Cunningham.
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The frequency of serum autoantibodies including anti-albumin antibodies was investigated for patients with various categories of alcoholic liver disease (ALD). The 95 patients were grouped into 3 categories: fatty liver (25 cases), alcoholic hepatitis (29 cases), and alcoholic cirrhosis (41 cases), and each group was matched with healthy controls by age, sex, ethnic origin and socio-economic status. For all patients with ALD, there was a 5-fold greater frequency over the controls of positive tests for antinuclear antibody (ANA), but titres were low; the pattern of ANA reactions was speckled in 50% of the cases. For patients with alcoholic cirrhosis, there was an 8-fold increase in frequency over the controls in anti-smooth muscle antibody (ASMA), but titres were low, pointing to a possibility that autoimmunity might be one of several determinants of progression of alcoholic hepatitis to cirrhosis. For none of the groups was there an increase over the controls in the mean titre of sheep red cell agglutinins, nor were there increased haemagglutinin titres of antibody to bovine serum albumin or to human albumin, arguing against a general increase in antibody production in ALD. Greater knowledge of the frequency, significance and pathogenicity of reactive autoantibodies which occur in response to various types of tissue damage is required for interpretation of the increase in ANA and ASMA in alcoholic liver disease.
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The digest of penicillopepsin (EC 3.4.23.7) with protease II from Myxobacter AL-1 gave five fragments which were separated on a Biogel P-100 column in 70% formic acid. The fragments were from 16 to 125 amino acids long. Two fragments were also isolated from a digest with a protease from Staphylococcus aureus. The analysis of these fragments by automatic sequencer gave a number of overlaps of the chymotryptic and thermolytic peptides. The available amino acid sequence data for penicillopepsin described in this paper and the accompanying papers (Kurosky, A. and Hofmann, T.:Can. J. Biochem. 54, 872 (1976);Rao, L and Hofmann, T.:Can. J. Biochem.54,885 (1976); Harris, C.I.,Rao, L., Shutsa, P., Kurosky, A. and Hofmann, T.: Can. J. Biochem. 54,895 (1976) have been combined and yield 15 fragments which range in lengths from 3 to 112 amino acid residues. These unique fragments account for virtually all the amino acids of the fungal protease. Four of the fragments with a total of 194 residues (about 60% of the molecule) have been aligned with corresponding sections of pig pepsin (EC 3.4.23.1) and with part of the N-terminal sequence available for calf chymosin (EC 3.4.23.4). In the alignments about 37% of the residues in the fungal enzyme are identical with at least one of the mammalian enzymes. An additional 20% are chemically similar. These results, together with previously reported active site directed modifications, show conclusively that penicillopepsin is an evolutionary homologue of the mammalian acid proteases.
A family of five individuals exposed to doves in their home were studied to evaluate possible factors leading to sympotmatic hypersensitivity pneumonitis documented in one member of the family, while the other exposed members remained asymptomatic. Antbodies against pigeon serum were domenstrated by radioimmunoassay in all subjects, and significant lymphocyte reactivity against pigeon antigens was demonstrable in three of the five members. The presence of serum antibody activity or lymphocyte reactivity against avian antigens or both did not differentiate the sick from the well family subjects. However, the possibility that increased exposure to antigen may correlate with higher degrees of antibody and lymphocyte reactivity as well as clinical disease is suggested from observation made in this family. No correlation was found between HL-A type and either symptomatic disease or immunological reactivity in these subjects.
A new, inexpensive device was developed for making individually prepared capsules that can be administered to experimental animals. The device was designed for accurate and rapid production of large numbers of various capsule sizes and drug dosages. During 12 months of usage, 21 capsules were prepared weekly for each of 44 dogs (924 total capsules) in approximately 2.5 work-hours/week. Each capsule contained a precise amount of drug to administer a specific dosage to each individual dog. No difficulties were encountered in the manufacture or utilization of this device, and it can be operated by untrained personnel.
An Aedes albopictus cell line was found contaminated with structures morphologically compatible with an alphavirus. Rapid isolation of a cytopathic virus was effected by combining sonication, concentration with Aquacide II-r, rate zonal sedimentation and subsequent plating of fractions on Vero cells under agar overlay. The virus caused neithedeath nor disease on inoculation into infant and adult mice. It produced a c.p.e. in Vero and BHK 21 cells, and multiplied in Singh's Aedes aegypti cells. The virus was identified serologically as chikungunya by complement-fixation and plaque reduction netralization test. Virus was not detected in a single attempt by these methods in the American Type Culture Collection A. albopicturs line. The presence of chikungunya virus in A. albopictus cells is not easily recognized and may complicate interpretation of experimental results.
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Two new methods are described for making chromosomal spreads of single antibody-forming cells. The first depends on the controlled rupture of cells in small microdroplets through the use of a mild detergent and application of a mechanical stress on the cell. The second is a microadaptation of the conventional Ford technique. Both methods have a success rate of over 50%, though the quality of chromosomal spreads obtained is generally not as good as with conventional methods. These techniques have been applied to an analysis of cell to cell interaction in adoptive immune responses, using the full syngeneic transfer system provided by the use of CBA and CBA/T6T6 donor-recipient combinations. When neonatally thymectomized mice were restored to adequate immune responsiveness to sheep erythrocytes by injections of either thymus cells or thoracic duct lymphocytes, it was shown that all the actual dividing antibody-forming cells were not of donor but of host origin. When lethally irradiated mice were injected with chromosomally marked but syngeneic mixtures of thymus and bone marrow cells, a rather feeble adoptive immune response ensued; all the antibody-forming cells identified were of bone marrow origin. When mixtures of bone marrow cells and thoracic duct lymphocytes were used, immune restoration was much more effective, and over three-quarters of the antibody-forming mitotic figures carried the bone marrow donor chromosomal marker. The results were deemed to be consistent with the conclusions derived in the previous paper of this series, namely that thymus contains some, but a small number only of antigen-reactive cells (ARC), bone marrow contains antibody-forming cell precursors (AFCP) but no ARC, and thoracic duct lymph contains both ARC and AFCP with a probable predominance of the former. A vigorous immune response to sheep erythrocytes probably requires a collaboration between the two cell lineages, involving proliferation first of the ARC and then of the AFCP. The results stressed that the use of large numbers of pure thoracic duct lymphocytes in adoptive transfer work could lead to good adoptive immune responses, but that such results should not be construed as evidence against cell collaboration hypotheses. Some possible further uses of single cell chromosome techniques were briefly discussed.
The effect of mutations that truncate the WT1 protein on in vitro hematopoietic differentiation from embryonal stem cells has been examined by CFU-A assay, o-dianisidine staining for heme, and RT-PCR analysis of the expression of fetal and adult globins. In two independently isolated ES cell lines the mutations delay but do not abolish hematopoiesis. Analysis of replated CFU-A colonies indicates that the delay occurs prior to the formation of hematopoietic stem cells. The results demonstrate a role for WT1 at the onset of hematopoiesis.
BACKGROUND: The purpose of this study was to examine the impact of fat reduction on vitamin and mineral density in the diets of groups of children who participated in the CATCH school-based intervention study for 2 1/2 years. METHODS: Twenty-four-hour dietary recalls were collected on 1,182 children residing in four states and with diverse ethnic backgrounds, at baseline when they were in third grade and again at follow-up when they were in fifth grade. RESULTS: With a 2.4% reduction in total fat intake from baseline levels in the intervention group, mean changes in vitamin and mineral intakes per 1,000 kcal of this group were small yet significant compared with the control group. As mean fat intake decreased, the nutrient density of most vitamins and minerals increased; the sole exceptions were vitamin E and sodium. Mean intake of all vitamins and minerals studied exceeded 2/3 of the RDA at both baseline and follow- up for both the intervention and the control groups. No significant differences were noted between treatment groups for the percentages of children meeting 2/3 or more of the RDA at both baseline and follow-up periods. CONCLUSIONS: CATCH was successful in maintaining mean vitamin and mineral intakes of intervention group children when total dietary fat was reduced.