Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Guidelines for selective radiological assessment of inversion ankle injuries.

A prospective study was performed to establish definitive guidelines for selective use of radiography in the assessment of inversion ankle injuries. Five hundred patients were included, representing 3.2% of the workload of the department during the study period. There were 379 soft tissue injuries, 56 malleolar fractures, 40 avulsion fractures, 21 fractures at the base of the fifth metatarsal, and four calcaneal fractures. Multiple logistic regression identified distal fibular tenderness, age, and ability to bear weight as the most important clinical variables in predicting important fractures (p less than 0.001). A policy of requesting x ray examination of only those patients with distal fibular tenderness or inability to bear weight or aged over 60, with a further proviso that no foot radiographs should be obtained, would produce a 60% reduction in ankle radiography in this centre without detriment to patient care.     

Occurrence of diphthamide in archaebacteria

We examined the nature of the diphtheria toxin fragment A recognition site in the protein synthesis translocating factor present in cell-free preparations from the archaebacteria Thermoplasma acidophilum and Halobacterium halobium. In agreement with earlier work (M. Kessel and F. Klink, Nature (London) 287:250-251, 1980), we found that extracts from these organisms contain a protein factor which is a substrate for the ADP-ribosylation reaction catalyzed by diphtheria toxin fragment A. However, the rate of the reaction was approximately 1,000 times slower than that typically observed with eucaryotic elongation factor 2. We also demonstrated the presence of diphthine (the deamidated form of diphthamide, i.e., 2-[3-carboxyamide-3-(trimethylammonio)propyl]histidine) in acid hydrolysates of H. halobium protein in amounts comparable to those found in hydrolysates of similar preparations from eucaryotic cells (Saccharomyces cerevisiae and HeLa). Diphthine could not be detected in hydrolysates of protein from the eubacterium Escherichia coli. Whereas both archaebacterial and eucaryotic elongation factors contain diphthamide, they differ importantly in other respects.     

Dehydrogenase activity and toxinogenesis of a production strain ofCorynebacterium diphtheriae during submerged cultivation

A simple biochemical procedure was obtained for studying metabolism ofCorynebacterium diphtheriae during submerged cultivation based on the modification of the assay of dehydrogenase activity using 2,3,5-triphenyltetrazolium chloride as redox indicator. Results obtained by the estimation of the dehydrogenase activity using TTC are in a good accordance with oxygen consumption assayed manometrically. By following dehydrogenase activity in submerged cultivations of a production strain ofCorynebacterium diphtheriae PW8-Weissensee we found that a massive toxin production is connected with the decrease of the activity of cells. This fall of activity occurs yet during the exponential phase of growth. Especially a sudden fall of succindehydrogenase activity exactly indicates the beginning of a considerable toxin accumulation in the medium. The presence of inhibitory concentrations of iron ions in the medium not only increases the level of dehydrogenase activity but changes its whole kinetics. A retarded and irregular fall of the activity occurs instead of a sharp one typical for good toxin production.     

Survival of Shigella in Sewage: II. Effect of Glycerol on Shigella flexneri and Shigella Bacteriophage1

Glycerol (30%) inhibited or delayed the adsorption of Shigella bacteriophage on its host organism, S. flexneri II; glycerol also inhibited or delayed the burst of phage, whether or not adsorption was carried out in the presence of glycerol. Studies of the mechanisms of these effects showed that viscosity and osmotic shock probably were not responsible for either phenomenon. The inhibition of adsorption, however, was proportional to the concentration of glycerol, and appeared to be a function of the hydroxyl groups on the glycerol molecule. The inhibition of burst seemed to be related to the osmotic pressure outside the bacterial cells.     

Epitopes recognized by the neutralizing antibodies of an HIV-1-infected individual.

Sera from individuals infected by HIV-1 usually neutralize multiple viral isolates. To determine the extent to which these neutralizing antibodies recognize a principal neutralizing determinant in the V3 region of the envelope protein gp120 (amino acids 308-332), one broadly neutralizing serum was fractionated by affinity chromatography on immobilized peptide columns. Antibodies that neutralize one isolate (HTLV-IIIMN) were substantially but not completely absorbed by the peptide corresponding to a portion of its V3 determinant, whereas the antibodies that neutralize two other isolates (HTLV-IIIB and HTLV-IIIRF) were not absorbed by homologous peptides corresponding to their neutralizing determinants. Neutralizing antibodies also failed to be absorbed by full length envelope protein gp160 and by two other envelope peptides previously reported to be broadly neutralizing epitopes (amino acids 254-274 and 735-752). We conclude that the infected individual had raised a type-restricted neutralizing response targeted at a linear epitope in the V3 region, and that broad neutralization resulted from recognition of epitopes not yet identified.