Sites of Tubulin Polymerization in PC 12 Cells

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.     

Pyruvate metabolism in mitochondria from cucumber cotyledons during early seedling development

Mitochondria were isolated from cucumber cotyledons during earlyseedling growth, and their capacity for pyruvate metabolisminvestigated. The rate of pyruvate oxidation was low. Evidenceis presented that suggests that this is due to low activitiesof the pyruvate transporter. Key words: Cotyledon, cucumber, germination, pyruvate oxidation     

The effects of vinblastine in assessment of the influence of age on proliferative activity of murine palate and footpad epithelium

Abstract
Data concerning changes in the rate of cell proliferation of stratified epithelia with increasing age are conflicting. In the present study young (3-month-old) and old (22-month-old) C57B1/6NNia male mice were injected intraperitoneally with 2, 3, 4 or 8 mg vinblastine sulfate/kg body weight and killed after 1.5, 3, 4.5 or 6 h. The number of arrested metaphase figures per 1000 basal cells was counted in histological sections. Data were analysed using a multivariate analysis of variance. There was a significant difference between the accumulation of mitotic figures in footpad epidermis and palate epithelium and both tissues contained an increased number of mitotic figures with increasing periods of accumulation at all dose levels. In the footpad epidermis neither the age of the animal nor the dose of vinblastine had a significant effect on the number of mitotic figures. In contrast, for palate epithelium the accumulation of mitotic figures was significantly less in the old mice compared with the young mice and at a dose of vinblastine of 2mg/kg compared with the higher doses. There was a statistically signifycant interaction between the dose of vinblastine and its period of action. It was concluded that the different tissues manifest a differential sensitivity to vinblastine and that only palate epithelium showed a significant reduction in proliferative activity with age.     

The efficacy of CGS 20267 in suppressing estrogen biosynthesis in patients with advanced stage breast cancer

The pharmacologic inhibition of aromatase activity has been the focus of clinical trials in patients with advanced stage breast cancer. Recent developments with imidazole compounds that inhibit aromatase activity suggest their clinical use as potent inhibitors of estrogen biosynthesis in postmenopausal breast cancer patients. In this Phase I, open-label, dose-range finding study, we examined the inhibitory potency of CGS 20267 on blood and urine levels of estradiol, estrone and estrone sulfate in 8 patients with metastatic breast cancer. Studies included evaluation of adrenal and thyroid function to look for evidence of general hydroxylase inhibition at dose levels effective for aromatase blockade. Patients were administered CGS 20267 at doses of 0.1 and 0.25 mg, once a day in ascending doses over a 12-week period. Preliminary data reveal that CGS 20267 elicits a striking suppression in plasma estradiol, estrone and estrone sulphate which was observed in some patients as quickly as within 24 h of the first dose. Estrogen suppression of over 90% was achieved within 2 weeks of therapy. No alterations in either baseline or ACTH (cortrosyn) stimulated cortisol and aldosterone levels were observed through the 12 weeks of therapy. In addition, 24 h urine sodium and potassium values were not appreciably altered during therapy. We conclude that CGS 20267 is a potent, specific inhibitor of estrogen biosynthesis in postmenopausal patients with metastatic breast cancer and effectively reduces blood and urine estrogens to undetectable levels.     

The mutation ofEscherichia coli to resistance to bacteriophages Tl and Tls

Summary The number of phage-resistant mutants in unirradiated and in ultraviolet-irradiated cultures ofE. coli strains B and B/r is usually greater when the selecting agent is Tls. It is shown that this cannot be explained by a) completely independent gene changes, b) the presence of host range phage mutants or c) new mutations due to prolonged survival of wild type bacteria in the prescence of phage. Evidence is presented which indicates that the difference in selection by Tl and Tls may be explained satisfactorily by the presence of incompletely expressed bacterial mutants in the cultures before plating. This constitutes indirect evidence for a phenotypic lag. It is shown further that the development of resistance to Tls probably precedes the development of full resistance to both phages.The appearance of new ultraviolet-induced mutations, selected by Tls, is found to fulfill expectations based on the existence of such a phenotypic lag.

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Zusammenfassung Die Anzahl der phagenresistenten Mutanten in unbestrahlten und UV-bestrahlten Kulturen derE. coli Stämme B und B/r ist im allgemeinen größer, wenn die Selektion mit Tls durchgeführt wird. Dieses kann nicht erklärt werden durch a) vollständig unabhängige Genveränderungen, b) die Gegenwart von hostrange Phagenmutanten, oder c) neue Mutationen bedingt durch längeres Überleben von Wildtypus-Bakterien in der Gegenwart von Phagen. Es wird gezeigt, daß der Unterschied in der Selektion von Tl und Tls ausreichend erklärt werden kann durch die Gegenwart von unvollständig manifestierten Bakterienmutanten, die vor dem plating in den Kulturen vorhanden sind. Dieses bildet indirekten Beweis für eine phänotypische Lagphase. Es wird weiterhin gezeigt, daß die Entwicklung der Resistenz gegenüber Tls wahrscheinlich der Entwicklung von Vollresistenz gegenüber beiden Phagen vorangeht.Das Auftreten von neuen UV-induzierten, durch Tls selektierten Mutanten stimmt mit dem Vorhandensein einer derartigen phänotypischen Lagphase überein.

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With 6 Figures in the text.