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.     

Eruptive epithelioid hemangioendothelioma with spindle cells

A 39-year-old white man presented with four discrete dermal nodules in his right upper arm. Biopsy revealed superficial dermal well-circumscribed nodules composed of solid areas and vascular spaces lined by epithelioid endothelial cells and a similar nodule composed of spindle and epithelioid cells. A moderate mitotic count of 3-4 mitoses/10 hpf was present. Multiple lesions erupted 1 month later distally and proximally to the original lesions. Magnetic resonance imaging of the right arm demonstrated a lesion in the humerus. Biopsy of the humerus showed a vascular tumor with similar histologic features to the overlying skin lesions. The differential diagnosis included epithelioid vascular tumors, bacillary angiomatosis, pyogenic granuloma, and Kaposi sarcoma. Vascular lesions containing epithelioid and spindle cells span a spectrum from benign to malignant. We believe these tumors belong in the category of hemangioendothelioma and propose the name eruptive epithelioid hemangioendothelioma with spindle cells. Our case emphasizes that eruptive cutaneous vascular lesions do not always suggest immunosuppression or malignancy. Additionally, it highlights the association between epithelioid vascular lesions of the skin and bone.     

Open versus closed epididymal sperm retrieval in men with secondarily obstructed vasal systems--a preliminary report

OBJECTIVE: To evaluate and compare sperm quality and suitability for intracytoplasmic sperm injection (ICSI) from open and percutaneous epididymal aspiration in men with obstructive azoospermia, and to determine the relevance of epididymal morphology. PATIENTS AND METHODS: A series of 20 men undergoing vasectomy reversal were evaluated by percutaneous (PESA) and open epididymal sperm aspiration (MESA) before undergoing surgery for reversal. Two samples were taken with PESA, one with the needle in situ (PESA1) and the second while withdrawing the needle (PESA2). Epididymal morphology was graded as normal, distended and grossly distended. Five men undergoing vasectomy served as a control, nonobstructed group for percutaneous aspiration. Analysis of the aspirates was performed immediately after operation with no knowledge of the treatment, and aspiration was considered successful if sperm suitable for ICSI were retrieved. RESULTS: In the obstructed group, 15 of 20 men had successful PESA and 13 of these also had successful MESA. PESA was successful bilaterally eight times compared with MESA on five occasions; two men with successful PESA had no success with MESA. PESA2 was five times more successful than PESA1. Only one PESA in the non-obstructed group was suitable for ICSI. PESA was successful in 21 of 25 distended or grossly distended epididymi compared with only three of 21 non-distended systems. CONCLUSION: PESA is a viable alternative to MESA in patients with obstructive azoospermia, particularly when associated with clinically distended epididymi.     

The genetic basis of epidermolysis bullosa simplex with mottled pigmentation

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins. Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two K5 alleles, leading to a Pro: Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals. Linkage analyses mapped the defect to this type II keratin gene (peak logarithm of odds score at phi = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type II keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.