Towards the physical map of the Trypanosoma cruzi nuclear genome: construction of YAC and BAC libraries of the reference clone T. cruzi CL-Brener

Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.     

Refining hollow obturator base using light-activated resin

Inclusion body myositis (IBM) is a separate class of the inflammatory myopathies with recently proposed clinical and pathological diagnostic criteria. An association between inflammatory myopathies and malignancy has been questioned in the literature. Recent reviews of the inflammatory myopathies suggest that only dermatomyositis is associated with malignancy. The largest study to date of patients with IBM found that 15% had a malignancy (6 of 40). We report the first documented case of IBM and concurrent transitional cell carcinoma of the bladder. We suggest that a causal relationship between IBM and malignancy may exist because of significantly improved functional strength gained after tumor removal.     

Neuronal-associated tumor necrosis factor (TNF alpha): its role in noradrenergic functioning and modification of its expression following antidepressant drug administration

Because of its wide variety of symptoms, the neurovascular compression syndrome of the upper thoracic aperture or thoracic outlet syndrome (TOS) has to be distinguished from several differential diagnoses, especially orthopedic ones. It is mainly characterized by pressure lesion of the brachial plexus and secondly, by accompanying vascular damages. An indication for surgery exists in cases of persisting or increasing complaints or function loss of shoulder, arm or hand muscles as well as in cases with occurrence of vascular damage. The treatment of choice consists of transaxillary resection of the first rib. This report presents a record of our results after transaxillary surgery and compares them to the results given in current international literature. During the last few years 3031 cases of transaxillary resections (13-473 cases) were described in international literature. The average follow-up period was 7.8 years (2-22 years). The results were good in 81.4% (50-93%) of the cases. From 02/80 to 03/94, we treated 67 patients with altogether 80 thoracic outlet syndromes (TOS). Each of them was treated similarly with extensive transaxillary resection of the first rib, excision of fibrous bands and, if necessary, of cervical ribs. Our results after surgery (84.5% of complete resolution or improvement of symptoms) compare favorably to those given by other authors. The average follow-up period was 6.6 years (0.5-14 years). Our results confirm, that transaxillary resection of the first rib is the method of choice in the treatment of thoracic outlet syndromes.     

Calicheamicin derivatives conjugated to monoclonal antibodies: determination of loading values and distributions by infrared and UV matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization mass spectrometry

Calicheamicin derivatives (MW approximately 1500) and monoclonal antibodies (MoAbs) conjugated to calicheamicin derivatives (MW approximately 150,000) were analyzed by UV-MALDI/MS, IR-MALDI/MS, and ESI/MS. These materials are potent anticancer agents. Calicheamicin derivatives and conjugates rapidly degrade upon UV irradiation but are relatively stable during IR irradiation and under ESI conditions. A unique feature of IR-MALDI/MS is a 2 times enhancement in resolution relative to UV-MALDI/MS for masses above approximately 50,000 Da resulting in a molecular ion envelope containing a series of partially resolved peaks of the calicheamicin-MoAb conjugates. The mass shift difference between the peak maxima corresponded to the mass change due to the covalent addition of calicheamicin derivatives to the monoclonal antibody. The distribution of the calicheamicin derivatives in the monoclonal antibodies was computed by deconvoluting the partially resolved peak envelope. A unique feature of the ESI mass spectra, under unit resolution conditions, is that the distribution of the carbohydrates can be well resolved for pure MoAbs and can be only partially resolved for conjugated MoAbs. Average loading values for calicheamicia derivatives when conjugated to MoAbs were computed from UV-MALDI/MS, IR-MALDI/MS, and ESI/MS data and the results compared with the average loading values obtained by UV absorption spectrometry. Very low average loading values were computed from UV-MALDI/MS data due to the degradation of the conjugated calicheamicin derivatives during the UV irradiation process. The IR-MALDI/MS average loading values, obtained with glycerol as the matrix, were consistent with the UV absorption spectrometry values for conjugates having hydrolytically stable linkers, but not when the linker contained a hydrolytically labile hydrazone. ESI/MS average loading values were generally lower than the corresponding values obtained by IR-MALDI/MS. The average loading values and distributions obtained using IR-MALDI/MS were more reliable than the corresponding ESI/MS values because the partially resolved, singly and doubly charged peaks in the IR-MALDI spectra can be mathematically deconvoluted, while the overlapping, highly multiply charged peaks of the electrospray spectra can only be partially deconvoluted.     

A comparison between emergency diagnostic and treatment unit and inpatient care in the management of acute asthma

BACKGROUND: Emergency diagnostic and treatment units (EDTUs) may provide an alternative to hospitalization for patients with reversible diseases, such as asthma, who fail to adequately respond to emergency department therapy. OBJECTIVE: To evaluate the medical and cost-effectiveness, patient satisfaction, and quality of life of patients receiving EDTU care for acute asthma compared with inpatient care. METHODS: A prospective, randomized clinical trial performed at 2 urban public hospitals enrolled patients with acute asthma (age range, 18-55 years) not meeting discharge criteria after 3 hours of emergency department therapy. Patients were treated with inhaled adrenergic agonists and steroids in an EDTU for up to 9 hours after randomization or with routine therapy in a hospital ward. Patients were followed up for 8 weeks. MAIN OUTCOME MEASURES: Discharge rate from the EDTU, length of stay, relapse rates, days missed from work or school, days incapacitated during waking hours, symptom-free days and nights, nocturnal awakenings, direct medical costs, patients satisfaction, and patient quality of life. RESULTS: The study consisted of 222 patients with asthma. Sixty-five patients (59%) treated in an EDTU were discharged home; the remainder were admitted to the hospital. There were no differences during the follow-up period in relapse rates (P = .74) or in any other morbidities between the EDTU and inpatient groups. There were significant differences in the length of stay, patient satisfaction, and quality of life favoring EDTU care. The mean (+/-SD) cost per patient in the EDTU group was $1202.79 +/- $1343.96, compared with $2247.32 +/- $1110.18 for the control group (P < .001). CONCLUSIONS: Treatment of selected patients with asthma in an EDTU results in the safe discharge of most such patients. This study suggests that quality gains and cost-effective measures can be achieved by the use of such units.     

Neurometabolism of active neuropsychiatric lupus determined with proton MR spectroscopy

PURPOSE: To determine the neurometabolism of patients with active neuropsychiatric systemic lupus erythematosus (NPSLE) by using proton MR spectroscopy. METHODS: Thirty-six patients with SLE and eight control subjects were studied with proton MR spectroscopy to measure brain metabolites. Peaks from N-acetylaspartate (NAA), creatine (Cr), choline (Cho), and at 1.3 parts per million (ppm) lipid, macromolecules, and lactate were measured. Patients were classified as having major NPSLE (seizures, psychosis, major cognitive dysfunction, delirium, stroke, or coma) (n = 15) or minor NPSLE (headache, minor affective disorder, or minor cognitive disorder) (n = 21). Patients with major NPSLE were severely ill and hospitalized. RESULTS: SLE patients had lower NAA and increased metabolites at 1.3 ppm than did control subjects (NAA/Cr(SLE) = 1.90 +/- 0.35, NAA/Cr(Control) = 2.16 +/- 0.26; 1.3 ppm/Cr(SLE) = 0.49 +/- 0.41, 1.3 ppm/Cr(Control) = 0.27 +/- 0.05). NAA/Cr in patients with current or prior major NPSLE was lower than in patients without major NPSLE. Increased peaks at 1.3 ppm were present in all SLE subgroups, but particularly in patients with major NPSLE. These resonances were not evident at an echo time of 136, indicating that these signals were not lactate. CONCLUSION: Major NPSLE, past or present, is associated with decreased levels of NAA. Elevated peaks around 1.3 ppm do not represent lactate even in severely ill patients, indicating that global ischemia is not characteristic of NPSLE. Neurochemical markers determined by MR spectroscopy may be useful for determining activity and degree of brain injury in NPSLE.     

Species dependence of enzyme-substrate encounter rates for triose phosphate isomerases

Triose phosphate isomerase (TIM) is a diffusion-controlled enzyme whose rate is limited by the diffusional encounter of the negatively charged substrate glyceraldehyde 3-phosphate (GAP) with the homodimeric enzyme's active sites. Translational and orientational steering of GAP toward the active sites by the electrostatic field of chicken muscle TIM has been observed in previous Brownian dynamics (BD) simulations. Here we report simulations of the association of GAP with TIMs from four species with net charges at pH 7 varying from -12e to +12e. Computed second-order rate constants are in good agreement with experimental data. The BD simulations and computation of average Boltzmann factors of substrate-protein interaction energies show that the protein electrostatic potential enhances the rates for all the enzymes. There is much less variation in the computed rates than might be expected on the basis of the net charges. Comparison of the electrostatic potentials by means of similarity indices shows that this is due to conservation of the local electrostatic potentials around the active sites which are the primary determinants of electrostatic steering of the substrate.