Peptidases in human bronchoalveolar lining fluid, macrophages, and epithelial cells: dipeptidyl (amino)peptidase IV, aminopeptidase N, and dipeptidyl (carboxy)peptidase (angiotensin-converting enzyme)

The modulation of proteolytic activity is an important factor in regulating the metabolism and function of peptide hormones. In this study, the activities of dipeptidyl (carboxy)peptidase (angiotensin-converting enzyme [ACE]), aminopeptidase N (APN), and dipeptidyl (amino)peptidase IV (DPP IV) were measured in the blood, the human bronchial epithelial and alveolar cells, bronchoalveolar macrophages, and the soluble phase of bronchoalveolar lavage (BAL) samples obtained from normal human volunteers and patients with pulmonary pathologic conditions. BAL fluid expressed ACE activity and very low levels of APN and DPP IV activities in the volunteer population, but higher levels could be measured in samples from patients. In patients, increased APN corresponded to a high granulocyte count, while DPP IV and ACE were associated with a high percentage of lymphocytes. Neither AIDS nor smoking induced an increased level of these enzymes. Immunohistochemical staining of bronchoalveolar smears with anti-human ACE monoclonal antibody showed that only macrophages expressed this enzyme. Enzyme histochemistry for DPP IV and APN showed that all leukocytes expressed these activities. APN, DPP IV, and ACE activities were also found in cell extracts of bronchoalveolar macrophages. In extracts of bronchial epithelial and alveolar cells, only APN and DPP IV activities were detected. Kinetic properties of the soluble enzymes in lavage supernatants were comparable to those of serum enzymes. These results demonstrate that soluble forms of cellular enzymes found in BAL fluid are regulated independently of blood and that different cell types may release these enzymes.     

Psychological and social predictors of motorcycle use by young adult males in New Zealand

Motorcycle riding is a significant cause of serious injuries to young males. Little is known about the psychological and social characteristics of these riders, despite such knowledge being potentially important for the targeting of appropriate injury prevention interventions. Using problem-behaviour theory to broadly guide and structure the research, the present study focused on identifying predictors of motorcycle riding. Previous research investigating differences between riders and non-riders has tended to be inconclusive, methodologically limited, and lacking in explicit theoretical foundations. The present research was based on the birth cohort enrolled in the Dunedin Multidisciplinary Health and Development Study (DMHDS), a comprehensive New Zealand longitudinal study of health, development, attitudes, and behaviours. Logistic regression models were built using prior measures of health risk behaviour, other psychological and social factors, and motorcycle riding history as potential predictors of any motorcycle use at the age of 18 years. The strongest predictors were early motorcycle riding, including illegal on-road driving at age 13 (OR 4.0; 95% CI 1.7, 9.1), below average reading skills (OR 2.4; 95% CI 1.3, 4.6) and fighting in a public place at age 15 (OR 2.9; 95% CI 1.2, 6.9). It was of particular interest that this profile tended to fit less well those subgroups of riders with greatest exposure to on-road motorcycle driving. Although based on small numbers, this finding was consistent with earlier cross-sectional research that linked casual and unlicensed driving with less protective motorcycling opinions and behaviours. Some implications for injury prevention and public policies regarding motorcycling are discussed. In particular, stricter enforcement of present licensing regulations and stronger penalties for their violation could help to reduce the number of less responsible riders.     

Determination of molecular-mass distribution of food-grade protein hydrolyzates by size-exclusion chromatography and chemiluminescent nitrogen detection

Size-exclusion chromatography (SEC) and chemiluminescent nitrogen detection (CLND) were used to estimate the molecular-mass distribution of food-grade protein hydrolyzates. Simultaneous CLND and UV (214 nm) detection is demonstrated for analytical SEC of an experimental casein hydrolyzate. In order to validate the estimated average M(r) values derived from the SEC column data, a preparative SEC separation of an extensive casein hydrolyzate was pursued. Fractions were collected on a time basis and analyzed by time-of-flight (TOF) mass spectrometry. A plot of TOF M(r) vs. SEC M(r) indicated that the peptides below M(r) of 1200 were eluted as estimated by the calibrated preparative SEC column. This paper demonstrates the power of using a dual CLND and UV detection system for analytical SEC analysis of protein hydrolyzates with a calibrated column.     

Characterization of enzymatic pectin digests by matrix-assisted laser desorption/ionization mass spectrometry

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the characterization of partially methyl-esterified enzymatic pectin digests is described. The sensitivities of several matrices, positive and negative ion modes and desalting techniques for these acidic oligosaccharides were compared. The most favorable results were obtained with a thin-layer preparation of a mixture of 2,4,6-trihydroxyacetophenone and nitrocellulose in the negative ion mode. Results are presented demonstrating the sensitive characterization of separated and unseparated high-ester pectin digests obtained after complete digestion using Aspergillus niger pectin lyase and the analysis of digests after chemical modification. In the case of unseparated digests, the analysis of methylation patterns is demonstrated. Oligomers with a degree of polymerization up to 40 were detected after enrichment of large oligomers by propan-2-ol precipitation.     

Pulmonary tuberculosis in Harare, Zimbabwe: analysis by spoligotyping

BACKGROUND: Over the last 10 years there has been a fourfold increase in cases of tuberculosis in Harare, Zimbabwe. The use of molecular epidemiology to understand tuberculosis transmission in this epidemic has been hampered by the availability of suitable culture facilities. A study was therefore undertaken to explore the potential of spoligotyping, a polymerase chain reaction based technique that does not require tuberculosis culture. METHODS: Adults attending a chest clinic with clinical or radiological pulmonary tuberculosis and one smear positive sputum were enrolled over one month. Demographic, socioeconomic, and clinical data were gathered using a standardised questionnaire. Molecular fingerprinting of genomic DNA recovered from sputum was performed by spoligotyping. RESULTS: Sixty one subjects (median age 28 years (range 18-73); 61% men) were recruited and 57 provided adequate sputum samples. Recent rural-urban migration or immigration was not common; 40% of subjects lived in crowded living conditions. DNA suitable for spoligotyping was recovered from 28 patients and 20 different genotypes of Mycobacterium tuberculosis were identified. Fifteen patients were infected with an M tuberculosis strain shared by one or more individuals. Patients infected with a shared spoligotype were not closely linked geographically within Harare, but were more likely to live in overcrowded conditions (69% versus 23%; odds ratio 6.85 (95% CI 1.2 to 47), p = 0.026). Analysis of the patients' original rural family homes revealed two geographically related spoligotype clusters. CONCLUSIONS: Spoligotyping may yield valuable molecular typing information in populations where tuberculosis culture is not available. This novel technique requires further development and evaluation in larger epidemiological studies.     

Pharmacokinetics and hypotensive effect of diltiazem in rabbits: comparison of diltiazem with its major metabolites

To assess the contribution of its metabolites to the antihypertensive effects of diltiazem, a previously established rabbit model has been used to compare the pharmacokinetics and haemodynamic effects of the drug with those of its major metabolites deacetyldiltiazem (M1) and deacetyl-N-monodemethyldiltiazem (M2). Diltiazem, M1 and M2 were administered separately to each animal (n = 5 or 6 per study group) as a single 5 mg kg(-1) intravenous dose. Blood samples, systolic and diastolic blood pressure (SBP and DBP) and heart rate were recorded for each rabbit up to 8 h, and urine samples were collected for 48 h post-dose. Plasma concentrations of diltiazem and its major metabolites were determined by HPLC. The results showed that systemic clearance (CL) and volume of distribution at steady state (Vdss) were smaller for diltiazem than for the metabolites. Diltiazem and the metabolites reduced both SBP and DBP, the effects of diltiazem being most potent. Their effects on heart rate were highly variable and not statistically different between treatment groups (P > 0.05). These results indicate that diltiazem is a more potent hypotensive agent than M1 or M2, possibly because of the higher plasma concentrations secondary to the smaller CL and Vdss of diltiazem compared with the metabolites. The effects of the metabolites might, however, be more sustained.