The pathway of leukemic cell death caused by glucocorticoid receptor fragment 465*.

The truncated glucocorticoid receptor mutant gene 465* codes for a protein that is interrupted by a frame-shift mutation in the second zinc finger of the natural DNA binding domain. Thus, 465* represents the natural amino acid sequence 1-465 followed by 21 novel amino acids starting at position 466. The entire ligand binding domain is missing. Prior studies have shown that transient transfection of the glucocorticoid-resistant leukemic T-cell clone ICR-27 with a plasmid expressing 465* rapidly reduces the number of viable cells. This response does not require activation by a steroid, and a hybrid protein consisting of green fluorescent protein fused to 465* is found primarily in the cytoplasm. In the present study, we present evidence that the decrease in cell number is due to a form of cell death that bears many of the classic characteristics of apoptosis. Expression of the 465* protein can be detected a few hours after electroporation and is followed by activation of caspase-3 as well as reduction of the mitochondrial inner transmembrane potential. The caspase-3 inhibitor ZVAD-fmk blocks 465*-dependent cell death when added acutely after electroporation, but fails to do so later. We conclude that the novel 465* gene causes cell death by apoptosis.

Involvement of caspases in 4-hydroxy-alkenal-induced apoptosis in human leukemic cells.

4-Hydroxynonenal (HNE), a reactive and cytotoxic end-product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death and in various cell types has been shown to induce apoptosis. We have demonstrated that HNE, at micromolar concentrations, induces dose- and time-dependent apoptosis in a leukemic cell line (CEM-C7). Interestingly, much higher concentrations of HNE (> 15-fold) were required to induce apoptosis in leukocytes obtained from normal individuals. We also demonstrate that HNE causes a decrease in clonogenicity of CEM-C7 cells. Furthermore, our data characterize the caspase cascade involved in HNE-induced apoptosis in CEM-C7 cells. Using specific fluorogenic substrates and irreversible peptide inhibitors, we demonstrate that caspase 2, caspase 3, and caspase 8 are involved in HNE-induced apoptosis, and that caspase 2 is the first initiator caspase that activates the executioner caspase 3, either directly or via activation of caspase 8. Our studies also suggest the involvement of another executioner caspase, which appears to be similar to caspase 8 but not caspases 2 and 3, in its specificity. The demonstration of decreased clonogenicity by HNE in the leukemic cells, and their higher susceptibility to HNE-induced apoptosis as compared to the normal cells, suggests that such compounds may have potential for leukemia chemotherapy.

Effect of aflatoxin G1 on germination, growth and metabolic activities of some crop plants.

The effect of different concentrations of aflatoxin G1 on growth and germination of Zea mays and Vicia faba seeds, as well as on some biochemical parameters viz chlorophyll, carotenoid, protein and lipid content of seedlings, were studied. Inhibition of seed germination and seedling growth of maize and broad bean increased with increases in toxin concentration. A reduction in carbohydrates in the shoot systems of maize and broad bean was accompanied by a corresponding reduction in chlorophyll content. The total proteins and total lipids of V. faba were significantly greater at a 10 micrograms/ml concentration of aflatoxin G1, whereas in Z. mays significant inhibition (p < 0.05) was observed. At 5.0 micrograms/ml aflatoxin G1 lipids and proteins were reduced in both plants but the effect was less obvious at lower concentrations.

Degradation of chicken feathers by Chrysosporium georgiae.

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin-salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8 pH at 30 degrees C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin-salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracellular keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones.

Glucocorticoid/oxysterol-induced DNA lysis in human leukemic cells.

Both glucocorticoids and oxysterols, steroids with quite different known transduction pathways, cause the death of lymphoid cells. Dual TUNEL/propidium iodide assays on sensitive human leukemic CEM-C7 clones treated with either steroid were clearly positive by 48 h, consistent with apoptosis. Both steroids evoked two distinctive types of DNA lysis: cleavage into large fragments of several different sizes and the classic "ladders", multiples of approximately 200 base pairs. Conventional gel electrophoresis showed that a small proportion of total DNA had undergone laddering 36-48 h after treatment with glucocorticoid or 24 h after oxysterol exposure. On field inversion gel electrophoresis of cellular DNA both steroids caused an increase in an array of large DNA fragments <50 kb in size. A 50 kb fragment appeared 36 h after treatment with either steroid, but only oxysterol treatment caused a significant increase in a 300 kb fragment. Oxysterol treatment did not result in DNA fragmentation in the resistant M10R5 subclone, which retained sensitivity to glucocorticoids. We conclude that glucocorticoids and oxysterols kill these cells with similar, but not identical, patterns of DNA lysis which occur just before or concomitant with the onset of cell death.

Keratinolytic fungi in Egyptian soils. I. Baited with hair and wool.

150 soil samples were collected, 90 from Nile Valley and Delta, 36 from desert and 24 from salt marshes. Human, buffalo and cow hair and sheep wool were used as baits at three incubation temperatures. Forty-four species which belong to twenty-one genera at 27 degrees C and forty-two which belong to twenty-two genera at 37 degrees C were collected. We isolated the following keratinophilic fungi Chrysosporium tropicum, C. keratinophilum, C. indicum, C. pannicola, C. queenslandicum, Trichophyton terrestre, T. mentagrophytes and Microsporum gypseum. Several other saprophytic fungi were isolated. No fungi were isolated at 45 degrees C.

Fungistatic action of natural oils and fatty acids on dermatophytic and saprophytic fungi.

Testing the inhibitory effects of some natural oils and fatty acids on growth of some of the dermatophytes revealed the high fungistatic effects of clove and peppermint oils. These oils when applied at 1% concentration with 1% tween 80 inhibited growth of the tested fungi seriously. Fatty acids were relatively uneffective and the possibility of including clove and peppermint oils in antidermatophytic drugs were suggested.