A 22-year-old man with sickle cell disease developed a conductive hearing loss as a result of extramedullary hematopoiesis in the right middle ear. This is the first reported case of conductive hearing loss from extramedullary hematopoiesis and only the second report of extramedullary hematopoiesis in the middle ear.

Adult↗

PubMedDate not supplied

IL2-ricin fusion toxin is selectively cytotoxic in vitro to IL2 receptor-bearing tumor cells.

Fusion toxins consist of peptide ligands linked through amide bonds to polypeptide toxins. The ligand directs the molecule to the surface of target cells and the toxin enters the cytosol and induces cell death. Ricin toxin is an excellent candidate for use in fusion toxins because of its extreme potency, the extensive knowledge of its atomic structure, and the years of experience with RTA chemical conjugates in clinical trials. We synthesized a baculovirus transfer vector with the polyhedrin promoter followed sequentially from the 5' end with DNA encoding the gp67A leader sequence, the tripeptide ADP, IL2, another ADP tripeptide, and RTB. Recombinant baculovirus was generated in Sf9 insect cells and used to infect Sf9 cells. Recombinant IL2-RTB protein was recovered at high yields from day 5 insect cell supernatants, partially purified by affinity chromatography, and characterized. The recombinant product was soluble and immunoreactive with antibodies to RTB and IL2, bound asialofetuin and lactose, and reassociated with RTA. In the presence of lactose to block galactose-binding sites on RTB, the IL2-RTB-RTA heterodimer was selectively cytotoxic to IL2 receptor, bearing cells. Specific cytotoxicity could be blocked with IL2. Thus, we report a novel targeted plant toxin fusion protein with full biological activity.

Animals↗

PubMedDate not supplied

Characterization of single site ricin toxin B chain mutants.

DNA encoding ricin B chain was modified by site-directed mutagenesis, and eight separate mutant RTB cDNAs including four novel mutants were ligated into the baculovirus transfer vector, pAcGP67A. Cotransfection of S. frugiperda Sf9 cells with BaculoGold DNA was followed by limiting dilution isolation of recombinant baculoviruses. Infection of Sf9 cells at a multiplicity of infection of 5 in the presence of 25 mM lactose produced 0.05-1 mg/L of soluble, glycosylated 34 kDa proteins immunoreactive with monoclonal and polyclonal antibodies to ricin B chain. Mutant ricin B chains were partially purified by monoclonal antibody immunoaffinity chromatography to 10-50% purity in near milligram quantities. The mutant ricin B chains had decreased lectin binding relative to plant ricin B chain as measured by binding to immobilized lactose and asialofetuin and cell binding immunofluorescence. The mutant ricin B chains reassociated with plant RTA similarly to plant RTB, and the recombinant heterodimers had slightly reduced cell cytotoxicity relative to ricin.

Animals↗

PubMedDate not supplied

Double-lectin site ricin B chain mutants expressed in insect cells have residual galactose binding: evidence for more than two lectin sites on the ricin toxin B chain.

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, contains a cell binding lectin subunit (RTB) disulfide linked to an RNA N-glycosidase protein synthesis-inactivating subunit (RTA). Investigations of the molecular nature of the lectin sites in RTB by X-ray crystallography, equilibrium dialysis, chemical modification, and mutational analysis have yielded conflicting results as to the number, location, and affinity of sugar-combining sites. An accurate assessment of the amino acid residues of RTB involved in galactose binding is needed both for correlating structure-function of a number of plant lectins and for the design and synthesis of targeted toxins for cancer and autoimmune disease therapy. We have performed oligonucleotide-directed mutagenesis on cDNA encoding RTB and expressed the mutant RTBs in insect cells. Partially purified recombinant proteins obtained from infected cell supernatants and cell extracts were characterized as to yields, immunoreactivities, asialofetuin binding, cell binding, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Two single-site mutants (subdomain 1 alpha or 2 gamma) and two double-site mutants (subdomains 1 alpha 2 gamma) were produced and studied. Yields varied by two logs with lower recoveries of double-site mutants. All the mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. Single-lectin site mutants displayed up to a 1 log decrease in asialofetuin binding avidity, while the double-site mutants showed close to a 2 log decrease in sugar binding. However, for each of the double-site mutants, residual sugar binding was demonstrated to both immobilized asialofetuin and cells, and this binding was specifically inhibitable with alpha-lactose. All mutants reassociated with RTA, and the mutant heterodimers were cytotoxic to mammalian cells with potencies 1000-fold or more times that of unreassociated wild-type RTA or RTB. These data support a model for three or more lectin binding subdomains in RTB.

Binding Sites↗

PubMedDate not supplied

Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking.

We performed genetic engineering to fuse enhanced green fluorescent protein (EGFP) to the N terminus of RTA, expressed the fusion protein in Escherichia coli, purified and reassociated EGFP-RTA with plant RTB, and purified EGFP-ricin by size exclusion HPLC. The fusion heterodimer was able to bind galactosides, intoxicate cells, and show strong fluorescence. Mammalian cells incubated with EGFP-ricin showed strong cell surface fluorescence at 4 degrees C and, on incubation at 37 degrees C, distributed initially to endosomes and then to Golgi vesicles. Variable sensitivity of mammalian cells to ricin and ricin fusion proteins may be due in part to different patterns of intracellular routing. Cells were incubated with ricin or EGFP-ricin, and inhibition of protein synthesis was measured. Human hepatocellular carcinoma Hep3B cells were 10-fold more sensitive to ricin and 85-fold more sensitive to EGFP-ricin than human epidermoid carcinoma KB cells. Epifluorescence microscopy of cells incubated with EGFP-ricin showed greater localization of the fluorescence signal in the Golgi compartments in Hep3B cells than in KB cells. These data support a model requiring a Golgi-dependent step in cell intoxication by ricin. The work further identifies the usefulness of green fluorescent protein fusions in the study of retrograde transport of internalized peptides.

Cell Survival↗

PubMedDate not supplied

Ejection fraction response of the left ventricle of the heart to acute cerebrovascular accident in patients with coronary artery disease.

The ejection fraction (EF) of the left ventricle was measured by radionuclide ventriculography in 64 patients during an acute cerebrovascular accident. Sixteen patients (12 with coronary artery disease) died within two weeks of the onset of symptoms and had only one EF measurement. In the remaining 48 patients, the EF was also measured two weeks and three months after the acute event. The ejection fraction of the patient who died soon after the acute stroke (52 +/- 18) was significantly lower than that of the patients who survived (64 +/- 10) (p less than 0.01). Of the patients who survived, 28 without history of coronary disease had an EF of 67 +/- 10 during the acute event. It was significantly higher than that measured after two weeks (60 +/- 10) p less than 0.01). In 10 patients with history of chronic stable angina pectoris, the EF (59 +/- 10) was significantly lower in the first study compared to that measured in the second (69 +/- 10) (p less than 0.02). Ten patients with no evidence of ischemia but with a history of myocardial infarction had a higher EF (61 +/- 11) during the first study as compared to the second (51 +/- 11) (p less than 0.05). In all patients there was no significant difference in the EF measurements between the second and the third study. It is suggested that the EF response of the left ventricle of the heart to the acute cerebrovascular accident is similar to that observed in a stress test.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMedDate not supplied

Splenic abscess.

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Abscess↗

PubMedDate not supplied

Sexual problems in rehabilitation.

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Female↗

PubMedDate not supplied

[Production of biologically active recombinant ricin B-chain].

Escherichia coli cells transformed with plasmids containing ricin B-chain coding sequences are shown to express this heterologous protein in inclusion bodies. After denaturation and renaturation of the product in the presence of glutathione and lactose, the recombinant ricin B-chain is soluble, biologically active and stable. Cytotoxicity of heterodimer containing this protein and ricin A-chain is found to be only ten times lower, than that of native ricin. Recombinant B-chain alone was nontoxic to cells (ID50 > 10(-6) M). Our data suggest that ricin B-chain oligosaccharides are essential for stability preserving protein from proteolytic degradation in cells.

Base Sequence↗