Fusion tyrosine kinases induce drug resistance by stimulation of homology-dependent recombination repair,prolongation of G(2)/M phase,and protection from apoptosis

Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGF beta R, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkin's lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G(2)/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G(2)/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G(2)/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.     

The Sequential Mechanism of Guanidine Hydrochloride-Induced Denaturation of cAMP Receptor Protein from Escherichia coli. A Fluorescent Study Using 8-Anilino-1-Naphthalenesulfonic Acid

cAMP receptor protein (CRP) regulates expression of a number of genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two structural domains. The biological activation of CRP upon cAMP binding may involve the subunit realignment as well as reorientation between the domains within each subunit. In order to study the interactions between the subunits or domains, we performed stopped-flow measurements of the guanidine hydrochloride (GuHCl)-induced denaturation of CRP. The changes in CRP structure induced by GuHCl were monitored using both intrinsic Trp fluorescence as well as the fluorescence of an extrinsic probe, 8-anilino-1-Naphthalenesulfonic acid (ANS). Results of CRP denaturation using Trp fluorescence detection are consistent with a two-step model [Malecki, and Wasylewski, (1997), Eur. J. Biochem. 243, 660], where the dissociation of dimer into subunits is followed by the monomer unfolding. The denaturation of CRP monitored by ANS fluorescence reveals the existence of two additional processes. One occurs before the dissociation of CRP into subunits, whereas the second takes place after the dissociation, but prior to proper subunit unfolding. These additional processes suggest that CRP denaturation is described by a more complicated mechanism than a simple three-state equilibrium and may involve additional changes in both inter- and intrasubunit interactions. We also report the effect of cAMP on the kinetics of CRP subunit unfolding and refolding.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.     

4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons

Primary spinal cord trauma can trigger a cascade of secondary processes leading to delayed and amplified injury to spinal cord neurons. Release of fatty acids, in particular arachidonic acid, from cell membranes is believed to contribute significantly to these events. Mechanisms of fatty acid-induced injury to spinal cord neurons may include lipid peroxidation. One of the major biologically active products of arachidonic acid peroxidation is 4-hydroxynonenal (HNE). The levels of HNE-protein conjugates in cultured spinal cord neurons increased in a dose-dependent manner after a 24-h exposure to arachidonic acid. To study cellular effects of HNE, spinal cord neurons were treated with different doses of HNE, and cellular oxidative stress, intracellular calcium, and cell viability were determined. A 3-h exposure to 10 microM HNE caused approximately 80% increase in oxidative stress and 30% elevation of intracellular calcium. Exposure of spinal cord neurons to HNE caused a dramatic loss of cellular viability, indicated by a dose-dependent decrease in MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium, inner salt] conversion. The cytotoxic effect of HNE was diminished by pretreating neurons with ebselen or N-acetylcysteine. These data support the hypothesis that formation of HNE may be responsible, at least in part, for the cytotoxic effects of membrane-released arachidonic acid to spinal cord neurons.     

Ligand-induced conformational and structural dynamics changes in Escherichia coli cyclic AMP receptor protein

Cyclic AMP receptor protein (CRP) regulates the expression of a large number of genes in E. coli. It is activated by cAMP binding, which leads to some yet undefined conformational changes. These changes do not involve significant redistribution of secondary structures. A potential mechanism of activation is a ligand-induced change in structural dynamics. Hence, the cAMP-mediated conformational and structural dynamics changes in the wild-type CRP were investigated using hydrogen-deuterium exchange and Fourier transform infrared spectroscopy. Upon cAMP binding, the two functional domains within the wild-type CRP undergo conformational and structural dynamics changes in two opposite directions. While the smaller DNA-binding domain becomes more flexible, the larger cAMP-binding domain shifts to a less dynamic conformation, evidenced by a faster and a slower amide H-D exchange, respectively. To a lesser extent, binding of cGMP, a nonfunctional analogue of cAMP, also stabilizes the cAMP-binding domain, but it fails to mimic the relaxation effect of cAMP on the DNA-binding domain. Despite changes in the conformation and structural dynamics, cAMP binding does not alter significantly the secondary structural composition of the wild-type CRP. The apparent difference between functional and nonfunctional analogues of cAMP is the ability of cAMP to effect an increase in the dynamic motions of the DNA binding domain.