Identification and quantitative studies of protein immobilization sites by stable isotope labeling and mass spectrometry

A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in (18)O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the (18)O/(16)O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher (18)O/(16)O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313, and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports.     

Global characterization of porcine intrauterine proteins during early pregnancy

Total protein secreted in the intrauterine lumen increases between day 10 and 13 post-estrus in both cyclic and pregnant gilts. The objective of this experiment was to identify those intrauterine proteins whose secretion changes during this time period. Sixteen mature gilts were either mated (day 0) or remained cyclic and were slaughtered at either day 10 or day 13 (n = 4 per status by day). At slaughter, each uterine horn was flushed with 20 ml Minimal Essential Medium. Flushings were dialyzed extensively against distilled water. A 0.5 ml aliquot of each was lyophilized, subjected to two-dimensional PAGE, and protein spots were identified following Coomassie staining of each gel. Densitometry was used to compare relative amounts of each spot. After statistical analysis, spots that differed due to either day, status, or day by status interaction were excised and digested in-gel with trypsin. The resulting peptides were analyzed by tandem mass spectrometry (MS/MS). Using MS/MS data, protein identification for each spot was attempted. There were 280 matching spots, of which 132 were significantly (P < 0.05 or 0.01) affected by pregnancy status, day, or the day by status interaction. Most (73%) spots increased from day 10 to day 13 with no effect of pregnancy. Several spots were identified as proteases or their inhibitors. Others potentially modify glycolipids and/or glycoproteins. These results indicate that the concentrations of many proteins within the intrauterine environment during early pregnancy are independent of the conceptus and could play roles in regulating the endometrial or conceptus glycocalyx.     

Cladribine (2-CDA) given as subcutaneous bolus injections is active in pretreated Waldenstr?m's macroglobulinaemia. Swiss Group for Clinical Cancer Research (SAKK)

Clinical trials with intravenous cladribine infusions in pretreated patients with Waldenstr?m's macroglobulinaemia have shown a response rate of 40%. Our pharmacokinetic studies revealed that the bioavailability of subcutaneous cladribine is complete but that the concentration-time profile is very different from intravenous administration. We designed this phase II multi-institutional trial to study the activity and toxicity of cladribine given as s.c. bolus injections in patients with symptomatic Waldenstr?m's macroglobulinaemia. Between May 1993 and October 1995, 25 patients were accrued: male/female 18/7, median age 65 years (range 44-85). All except one patient had been pretreated with more than one regimen (median 2, range 0-10). 18 patients had progressed under previous therapy and six were in relapse. All patients received cladribine for a total dose of 0.5 mg/kg per cycle as s.c. bolus injections divided over 5 d at > or = 4 week intervals, for a maximum of six cycles. All 25 patients were evaluable for toxicity and response. A total of 67 cycles were administered (median 3 cycles, range 1-6). Overall response rate including disease stabilization which had been progressive under previous therapy was 68%. 10 patients (40%, 95% CI 21-61%) achieved a partial remission. Seven responders had been progressive under previous therapy. Maximum responses were reached no later than the third cycle. Median time to treatment failure and remission duration were 4.4 (range 0.5-33) and 8 months (5-29), respectively. Four patients (16%) suffered from infections W.H.O. grade > or = 2 (pneumonia grade 2, Staphylococcus septicaemia grade 3, viral encephalitis and pneumonia, both grade 4 with complete resolution). No other severe adverse events were observed. Cladribine given as s.c. 5 d bolus injections was found to be active in pretreated Waldenstr?m's macroglobulinaemia and resulted in durable remissions.     

Arm work interferes with normal ventilation

Arm work, by limiting movement of the chest wall and use of the respiratory muscles, may alter breathing pattern and gas exchange sufficiently to interfere with the ability to perform certain tasks. To determine the effects of arm work on breathing pattern during a well-controlled work task, depth of breathing, breathing frequency and end-expiratory lung volume (EELV) were measured at rest and during cycling exercise using an arm and a leg ergometer. Six subjects performed arm work at light, moderate and heavy intensities (30%, 60% and 90% of maximum arm work capacity respectively) and leg work at three intensities where ventilation was matched for that achieved during the arm work. This matching was necessary since the level of ventilation affects the breathing pattern. Subjects breathed on a mouthpiece and tubing that led to automated equipment for the measurement of respiratory variables. Ventilation during arm work was accomplished with a lower depth of breathing, a higher breathing frequency and a decreased EELV compared to leg work. Arm work places increased demands on the ventilatory system, including the muscles of respiration that are also recruited for task performance. The competition for using these muscles for breathing as opposed to a particular work task may result in a compromise in breathing capacity that ultimately may limit the ability to perform tasks requiring sustained heavy use of the arms. These increased demands on the upper body muscles must be considered when evaluating the ability of individuals to perform tasks that involve heavy arm work.     

Inhibition of endive (Cichorium endivia L.) polyphenoloxidase by a Carica papaya latex preparation

When endive polyphenoloxidase (PPO) was incubated with a crude papaya latex extract, it rapidly lost its activity. Inactivation was ascribed to thermostable nonenzymatic factors of low molecular weight. These factors were partially purified by a two step protocol including gel filtration chromatography on Biogel P2 and ion exchange chromatography using DEAE Sephadex A25. The PPO-inactivation rate was first order, when either inactivating agent or proton concentration was evaluated. Inactivation could be partially reversed by CuSO₄, which suggested that the inactivating factor(s) bound to the copper site of the enzyme. On a more rapid time scale than inactivation, papaya latex extract acted also as a weak noncompetitive PPO inhibitor.     

Structural analysis of peptide substrates for mucin-type O-glycosylation

The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.     

Composition and oxidative stability of hydro‐cracked base oils and comparison with a PAO

The oxidative stability of nine hydrocracked base oils from seven producers and a PAO was compared using the standard IP 48 test. After a comprehensive characterisation of the oils, an evaluation of oxidative stability was carried out by measuring some common oil parameters, such as viscosity characteristics, carbon residue, pentane insolubles, and acid number. Additionally, the compound‐class composition of the fresh and oxidised oils was determined, and an FTIR spectroscopic analysis was carried out. The oxidative stability of the hydrocracked oils was largely affected by the sulphur and aromatic hydrocarbon concentration in the oils. Oils with an increased sulphur content (above 80 ppm) had better oxidative stability than oils with a low sulphur content (20 ppm and lower), and there was a relatively large variability in the stability of the oils depending on the sulphur concentration. The oxidative stability of most of the hydrocracked oils with a low sulphur content was similar, and matched somewhat the stability of the polyalphaolefin.     

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions

Phytosterols play essential roles in many plant cell mechanisms. They are of industrial interest since, as part of the diet, they can reduce low density lipoprotein cholesterol. An increase in plant sterol contents, by improved crop varieties or crop management, could help to answer industrial demands and also to develop environmentally friendly extraction methods. The aim of this study was to evaluate genotypic variability of sterol content in cultivated sunflower and, in particular, effects of sowing date. Results showed large variability among a collection of sixteen sunflower inbred lines and hybrids. Total sterols varied almost twofold between extreme genotypes. A delay of sowing, giving higher temperatures during seed formation, induced a general increase in total sterol concentration by up to 35%, as well as variation in sterol composition according to genotype. These results are considered with an aim of improving sterol content by sunflower breeding programmes.