Rapid tryptic mapping using enzymatically active mass spectrometer probe tips

A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient.     

Radiation dose in critical organs due to non-coplanar irradiation of the hypophysis

BACKGROUND: In order to estimate the somatic and genetic risk associated with a non-coplanar linac-based radiation technique of the pituitary gland, systematic secondary-dose measurements in a phantom and sample measurements of the dose near critical organs of patients were performed. PATIENTS AND METHODS: For measurements of the dose outside the primary radiation field an acrylic-PVC phantom was used which was irradiated with a single field (4 x 4 cm2). Eight patients with pituitary tumors were treated isocentrically with a combination of sagittal and transverse rotational arcs. To measure the dose in critical organs. LiF thermoluminescence dosimeters (TLD) in chip form were placed onto 1 eyelid, the skin over the thyroid, and the patient's clothes covering the region of breasts and ovaries of female patients and the testicles of male patients. Measurements were performed for all patients during 1 sagittal irradiation and for the majority of patients during 1 transverse irradiation. RESULTS: The phantom measurements demonstrated that the secondary dose measured on the patients surface can be considered as a good approximation for the dose in adjacent organs. The median dose in critical organs for sagittal irradiation was in the range of 25.8 mGy (eyes) to 1.9 mGy (testicles), and for transverse irradiation in the range of 23.3 mGy (eyes) to 1.3 mGy (testicles). The ratio of median organ doses for sagittal and transverse irradiation was 2.1 for the thyroid gland, 1.1 for the eyes, and 1.5 for the other organs. CONCLUSIONS: The dose in critical organs due to non-coplanar irradiation of the pituitary gland is only a small fraction of the dose delivered to the reference point of the planning target volume. The risk of a radiation-induced tumor and a genetic consequence associated with these small doses is generally less than 1% and 0.1%, respectively.     

A randomized prospective trial of the obstetric forceps versus the M-cup vacuum extractor

OBJECTIVE: Our purpose was to determine the efficacy of the obstetric forceps versus the M-cup, a new vacuum extractor cup, and maternal-neonatal complication rates. STUDY DESIGN: Over a 10-month period operative vaginal deliveries were randomized between the obstetric forceps and the M-cup vacuum extractor cup. Maternal demographics, indication for intervention, analgesia, position, station, degree of asynclitism, fetal caput-molding, and time from application to delivery were prospectively recorded. Episiotomy and extensions, lacerations, and the reason for abandonment of the randomized instrument were noted in both groups. Fetal weight, Apgar scores, cord arterial gases, hyperbilirubinemia, phototherapy, and any evidence of fetal trauma were documented at delivery or in the nursery. RESULTS: Six hundred thirty-seven women were randomized, 315 in the forceps group and 322 in the M-cup group. There were no differences in maternal demographic variables. The station, position, degree of asynclitism, or requirement for rotation was not different between the groups. The corrected efficacy rates were forceps 92% and M-cup 94% (p = 0.217). The M-cup deliveries were accomplished more rapidly than forceps deliveries (p < 0.001) and were associated with a lower rate of episiotomy (p < 0.001), third-degree (p < 0.001) and fourth-degree (p = 0.002) lacerations, but blood loss as clinically estimated (p = 0.232) or as measured by hemoglobin levels (p = 0.166) was not significantly different. Forceps deliveries were associated with fewer clinically diagnosed cephalhematomas (p = 0.015) than M-cup deliveries were, but there were no differences in the number of neonates diagnosed with hyperbilirubinemia (p = 0.377) or in the number of infants treated with phototherapy (p = 0.660). CONCLUSIONS: The M-cup vacuum extractor cup appears to be as efficient (and faster) than the obstetric forceps but is associated with significantly more fetal cephalhematomas, whereas maternal injuries are more common with the forceps.     

Structural requirements of synthetic and natural product lipo-chitin oligosaccharides for induction of nodule primordia on Glycine soja

Rhizobia synthesize a class of lipo-chitin oligosaccharides that induce root hair deformation and induce the initiation of nodule structures on legume roots. These lipo-chitin oligosaccharides are tetra- and penta-lipo-oligosaccharides of N-acetylglucosamine with an acyl substitution on the nonreducing end and are commonly known as Nod factors. In this study, we demonstrate that synthetic analogs of natural product Nod factors have the same biological activities. To determine structure-activity relationships, a collection of synthetic and natural product lipo-chitin oligosaccharides was assayed on Glycine soja. All biologically active lipo-chitin oligosaccharides induced both root hair deformation and nodule initiations on G. soja. The most active lipo-chitin oligosaccharides deformed root hairs at 10(-15) M and induced nodules at 1 ng of lipo-chitin oligosaccharide per spot inoculation. Plant responses demonstrate an interdependence of backbone length and the presence of substitutions on the reducing end. Lipo-chitin oligosaccharides containing four N-acetylglucosamine residues were active only without a reducing end modification, whereas lipo-chitin oligosaccharides containing five N-acetylglucosamine residues were active only with reducing end modification. The plant thus recognizes lipo-chitin oligosaccharides without reducing end substitutions despite the importance of these modifications for host range.     

Artificial neural networks and logistic regression as tools for prediction of survival in patients with Stages I and II non-small cell lung cancer

BACKGROUND: This study examines the role of participation in psychosocial treatment as a mediator of the clinical effectiveness of clozapine. METHODS: Subjects participated in a 12-month double-blind random-assignment trial comparing clozapine and haloperidol in patients hospitalized 30 to 364 days for refractory schizophrenia at 15 Department of Veterans Affairs medical centers. A broker-advocate case management intervention was used to facilitate participation in psychosocial treatments and to document such participation. RESULTS: Between those who continued receiving clozapine (n=122) or a conventional antipsychotic drug (n=169) for 12 months, those receiving clozapine were more likely to participate in psychosocial rehabilitation treatment. Although they were no more likely to receive clinical recommendations for such treatments, they were more likely to both verbally accept recommendations and to act on them. Structural equation modeling shows that participation in psychosocial treatment did not play a mediating role in clozapine's effect on outcomes at 6 months, but was associated with both reduced symptoms and improved quality of life at 12 months. CONCLUSIONS: Clozapine facilitates participation in psychosocial treatment, and such enhanced participation is associated with improved quality-of-life and symptom outcomes. Psychosocial rehabilitation should be offered concomitantly with clozapine.     

Risk factors for avascular bone necrosis in systemic lupus erythematosus

OBJECTIVE: To study the predictive factors for avascular necrosis (AVN) of bone in patients with systemic lupus erythematosus (SLE). METHOD: The records of 38 SLE patients who developed clinically apparent AVN during the course of their disease were reviewed. Information on clinical presentation, corticosteroid usage and autoantibody profiles was obtained, and comparison was made between these patients and 143 consecutive control SLE patients who did not have AVN. RESULTS: The point prevalence of AVN in our SLE population was 12%. Patients with AVN, when compared with controls, had a significantly higher incidence of neurological disease (39% vs 14%; P < 0.001) and Cushingoid body habitus after steroid treatment (79% vs 53%; P = 0.004). The highest cumulative prednisolone dose in 1 and 4 months was significantly higher in the AVN group than the controls (1.8 vs 1.1 and 4.5 vs 2.8 g, respectively; P < 0.01 in both) and showed a linear trend with the incidence of AVN (chi2 test for trend, P < 0.01 in both). Lupus anticoagulant was associated with AVN (P = 0.02, odds ratio 2.88 [1.14-7.28]). Logistic regression analysis revealed that the highest cumulative prednisolone dose administered in 4 months, the maximum and mean daily prednisolone dosage, and the lupus anticoagulant were independent risk factors for AVN. CONCLUSIONS: Corticosteroid remains the major predisposing factor for AVN in SLE. Patients who require an initial high-dose steroid for disease control are at risk of AVN, especially if they are positive for the lupus anticoagulant or develop Cushingoid habitus after steroid treatment. High-risk patients should be closely monitored so that early AVN can be diagnosed by sensitive techniques such as magnetic resonance imaging and radioisotope bone scanning.     

Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils

BACKGROUND: As assessed by flow cytometry, the increase in hydrogen peroxide in individual neutrophils from old volunteers was significantly greater than in neutrophils from young volunteers. To explain the discrepancy in previous reports that showed reduced superoxide generation with age and our finding, we measured the kinetics of antioxidative enzymes. METHODS: Neutrophils were obtained from young (ages 21-34) and old (ages over 65) volunteers. The increase in hydrogen peroxide following stimulation with formyl peptide in individual neutrophils was assessed by flow cytometry by using dihydrorhodamine 123. The enzyme kinetics was determined from the best fit curve using Michaelis-Menten equations. RESULTS: Aging was associated with a significant reduction in the Vmax for glutathione peroxidase. The decreased activity was not due to selenium deficiency as the serum and neutrophil concentrations were identical with age. Following activation, a significant increase in the Km was noted in neutrophils from young but not from old volunteers. CONCLUSIONS: These results account for the increased intracellular accumulation of hydrogen peroxide as a function of age in stimulated neutrophils. These results provide evidence in humans of an age-related impairment in antioxidative defense mechanisms that support the free radical theory of aging.     

Skeletal myogenesis on elastomeric substrates: implications for tissue engineering

Studies geared towards understanding the interaction between skeletal muscle and biomaterials may provide useful information for the development of various emerging technologies, ranging from novel delivery vehicles for genetically modified cells to fully functional skeletal muscle tissue. To determine the utility of elastomeric materials as substrates for such applications, we asked whether skeletal myogenesis would be supported on a commercially available polyurethane, Tecoflex SG-80A. G8 skeletal myoblasts were cultured on Tecoflex two-dimensional solid thin films fabricated by a spin-casting method. Myoblasts attached, proliferated, displayed migratory activity and differentiated into multinucleated myotubes which expressed myosin heavy chain on solid thin films indicating that Tecoflex SG-80A was permissive for skeletal myogenesis. Porous three-dimensional (3-D) cell scaffolds were fabricated in a variety of shapes, thicknesses, and porosities by an immersion precipitation method, and where subsequently characterized with microscopic and mechanical methods. Mechanical analysis revealed that the constructs were elastomeric, recovering their original length following 100% elongation. The 3-D substrates were seeded with muscle precursors to determine if muscle differentiation could be obtained within the porous network of the fabricated constructs. Following several weeks in culture, histological studies revealed the presence of multinucleated myotubes within the elastomeric material. In addition, immunohistochemical analysis indicated that the myotubes expressed the myosin heavy chain protein suggesting that the myotubes had reached a state of terminal differentiation. Together the results of the study suggest that it is indeed feasible to engineer bioartificial systems consisting of skeletal muscle cultivated on a 3-D elastomeric substrate.