Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins

The coding sequence for human serum transferrin was assembled from restriction fragments derived from a full-length cDNA clone isolated from a human liver cDNA library. The assembled clone was inserted into the expression vector pNUT and stably transfected into transformed baby hamster kidney (BHK) cells, leading to secretion of up to 125 mg/L recombinant protein into the tissue culture medium. As judged by mobility on NaDodSO4-PAGE, immunoreactivity, spectral properties (indicative of correct folding and iron binding), and the ability to bind to receptors on a human cell line, initial studies showed that the recombinant transferrin, is identical to three commercial human serum transferrin samples. Electrospray mass spectrometry (ESMS), anion-exchange chromatography, and urea gel analysis showed that the recombinant protein has an extremely complex carbohydrate pattern with 16 separate masses ranging from 78,833 to 80,802 daltons. Mutation of the two asparagine carbohydrate linkage sites to aspartic acid residues led to the expression and secretion of up to 25 mg/L nonglycosylated transferrin. ESMS, anion-exchange chromatography, and urea gel analysis showed a single molecular species that was consistent with the expected theoretical mass of 75,143 daltons. In equilibrium binding experiments, the nonglycosylated mutant bound to HeLa S3 cells with the same avidity and to the same extent as the glycosylated protein and the three commercial samples. These studies demonstrate conclusively that carbohydrate has no role in this function.     

Reversal of chloroquine resistance in murine malaria parasites by prostaglandin derivatives

An oligomeric ester of prostaglandin B2 (OC-5186) was found to reverse chloroquine resistance in the murine malarial parasite Plasmodium berghei. When mice were infected with either chloroquine-sensitive or -resistant P. berghei on day 0 (by intraperitoneal injection of 1 x 10(6) parasitized erythrocytes), they died before day 23. When treated with 15 mg/kg/day of chloroquine for the first four days of infection, all mice infected with the sensitive-strain survived, while all those infected with the resistant strain died before day 23. When OC-5186 (3-12 mg/kg/day) was administered in combination with chloroquine for the first four days, 60% of the animals infected with the resistant strain survived. The differences in the survival rate between the group treated with chloroquine only and the group treated with a combination of drugs (chloroquine plus 3-12 mg/kg/day of OC-5186) were significant. There was also a significant inhibition of parasitemia in the group treated with the combination of drugs. The combinations of chloroquine and a monomer ester of prostaglandin B2 (OC-5181) had some antimalarial activity, but the differences between the chloroquine-treated group and the combination treatment group were not significant in terms of both the parasitemia and the survival rate. Another oligomeric ester of prostaglandin E1 (MR-356) as well as unesterified monomer prostaglandins (PGA2 and PGB2) were ineffective by themselves and in combination with chloroquine.     

Inversion polymorphism in natural populations of Drosophila bipectinata

Inversion polymorphism was studied in seven natural populations of Drosophila bipectinata, five from northern and two from southern India. Chromosomal analysis of these populations revealed the presence of three paracentric inversions which are widespread in populations of D. bipectinata. Quantitative data indicated that the frequency of inversions and the level of inversion heterozygosity were very low in populations of D. bipectinata. There is no evidence for genetic differentiation between populations as a result of inversion polymorphism. These findings provide evidence for rigid chromosomal polymorphism in D. bipectinata. Significant non-random associations between inversions indicated epistatic interaction between inversions in natural populations of D. bipectinata.     

Partially glucose-capped oligosaccharides are found on the hemoglobins of the deep-sea tube worm Riftia pachyptila

We report here the structural determination of N-linked oligosaccharides found on extracellular hemoglobins of the hydrothermal vent tube worm Riftia pachyptila. Structures were elucidated by a combination of electrospray ionization tandem mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry, normal-phase high performance liquid chromatography, and exoglycosidase digestion. The sugar chains were found to consist mainly of high-mannose-type glycans with some structures partially capped by one or two terminal glucose residues. The present study represents the first report of the occurrence of glucose capping of N-linked carbohydrates in a secreted glycoprotein of a metazoan. Previously, glucose capping has only been described for a membrane-bound surface glycoprotein from the unicellular parasite Leishmania mexicana amazonensis.     

A trial of the long-term use of Sedacorone (amiodarone) under polyclinic conditions for the prevention of paroxysms of atrial fibrillation

Sedacorone (amiodarone) was given to 70 outpatients with coronary heart disease for 1.5-2 years to prevent paroxysms of cardiac arrhythmia after recovery of sinus rhythm. Sedacorone was administered initially in the dose 600 mg/day for 10-12 days, then the dose lowering was adjusted to the tolerance, heart rate and ECG readings. The minimal dose of 200 mg/day was taken for 5 days a week with a 2-day interval. In adequate individual dose Sedacorone prevented paroxysms of cardiac fibrillation or made the paroxysms less frequent in 91.4% of patients. Careful selection of patients and regular control helped avoid serious cardial and extracardiac complications. Sedacorone is recommended as a first-line medicine for outpatients with frequent paroxysms of cardiac fibrillations after recovery of the sinus rhythm to prevent the paroxysms recurrences.     

Quantification of information transfer via cellular signal transduction pathways

OBJECTIVE: Symptomatic malignant pleural effusions are common sequelae in patients with certain malignancies. Pleurodesis via bedside thoracostomy is the current treatment option most commonly used. To our knowledge, this is the first prospective randomized trial to examine which agent, bleomycin or talc slurry, is superior in terms of effectiveness, safety, and cost. PATIENTS AND METHODS: Between July 1992 and March 1995, 35 patients presenting to our medical center with symptomatic malignant pleural effusions were prospectively randomized to undergo chemical pleurodesis with either bleomycin or talc slurry via bedside thoracostomy. The conditions of patients were assessed and graded before and after treatment concerning pain, dyspnea, and chest radiographs. RESULTS: Twenty-nine patients who underwent 33 treatments (14 with bleomycin and 19 with talc) were available for follow-up. Follow-up ranged from 2 weeks to 8 months (mean, 1.7 months). Both groups demonstrated notable improvement in both pain and dyspnea following treatment, but there were no statistically significant differences between groups in the amount of improvement (two-tailed Student's t test). Permanent control of effusions, defined objectively on chest radiograph, was achieved with 11 bleomycin treatments (79%) and 17 talc treatments (90%) (p=0.388). The procedures were well tolerated and no significant adverse effects were observed. Talc is a much less costly agent than bleomycin ($12.36 cost to our medical center per treatment for talc vs $955.83 for bleomycin). CONCLUSION: Given the similar efficacy and significant cost advantage, we conclude that talc is the agent of choice when utilizing pleurodesis for control of symptomatic malignant pleural effusions.     

Validation of computerized Swedish dog and cat insurance data against veterinary practice records

Spontaneous cellular reorganisation at the lesion site has been investigated following massive spinal cord compression injury in adult rats. By 2 days post operation (p.o.), haemorrhagic necrosis, widespread axonal degeneration, and infiltration by polymorphnuclear granulocytes and OX42-positive macrophages were observed in the lesion site. By 7 days p.o., low affinity nerve growth factor receptor-positive Schwann cells, from activated spinal roots, were identified as they migrated far into the lesion. Between 7 and 14 days p.o., the overlapping processes of Schwann cells within the macrophage-filled lesion formed a glial framework which was associated with extensive longitudinally orientated ingrowth by many neurofilament-positive axons. Relatively few of these axons were calcitonin gene-related peptide (CGRP)-, substance P (SP)-, or serotonin (5HT)-positive; however, many were glycinergic or gamma aminobutyric acid (GABA)ergic. At 21 and 28 days p.o. (the longest survival times studied), a reduced but still substantial amount of orientated Schwann cells and axons could be detected at distances of up to 5 mm within the lesion. Glial fibrillary acidic protein (GFAP) immunoreactivity demonstrated the slow formation of astrocytic scarring which only became apparent at the lesion interface between 21 and 28 days p.o. The current data suggest the possibility of developing future therapeutic strategies designed to maintain or even enhance these spontaneous and orientated regenerative events.