Identification and characterization of genes required for hyphal morphogenesis in the filamentous fungus Aspergillus nidulans.
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In the filamentous fungus Aspergillus nidulans, germination of an asexual conidiospore results in the formation of a hyphal cell. A key feature of spore germination is the switch from isotropic spore expansion to polarized apical growth. Here, temperature-sensitive mutations are used to characterize the roles of five genes (sepA, hypA, podB-podD) in the establishment and maintenance of hyphal polarity. Evidence that suggests that the hypA, podB, and sepA genes are required for multiple aspects of hyphal morphogenesis is presented. Notably, podB and sepA are needed for organization of the cytoskeleton at sites of polarized growth. In contrast, podC and podD encode proteins that appear to be specifically required for the establishment of hyphal polarity during spore germination. The role of sepA and the pod genes in controlling the spatial pattern of polarized morphogenesis in germinating spores is also described. Results obtained from these experiments indicate that the normal pattern of germ-tube emergence is dependent upon the integrity of the actin cytoskeleton. (+info)
The effect of mannitol versus dimethyl thiourea at attenuating ischemia/reperfusion-induced injury to skeletal muscle.
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OBJECTIVE: Mannitol is used as a treatment for skeletal muscle ischemia/reperfusion (I/R) injury in humans, despite the fact that its effectiveness in vivo is still disputed. The purpose of this study was to determine the efficacy of mannitol in attenuating I/R injury at the microcirculatory level. METHODS: The study was designed as an experimental study with male Wistar rats. The main outcome measures were intravital microscopy, which was used to measure capillary perfusion, capillary and venular red blood cell velocity (VRBC), and leukocyte-endothelial interactions in the extensor digitorum longus muscle of the rat hind limb before and after ischemia. In addition, tissue injury was assessed during reperfusion with the fluorescent vital dyes bisbenzimide and ethidium bromide. Dimethyl thiourea (DMTU), a highly effective therapeutic agent of experimental I/R injury, was used as a positive control. RESULTS: No-flow ischemia (2 hour) resulted in a 40% drop in capillary perfusion, a decline in capillary and venular VRBC, and increased leukocyte venular adherence and tissue infiltration. Tissue injury increased to a constant level during reperfusion. Mannitol attenuated capillary malperfusion during the first 60 minutes of reperfusion and prevented a decline in capillary VRBC. However, mannitol did not reduce tissue injury or leukocyte adherence and infiltration during reperfusion. By comparison, DMTU not only prevented the perfusion deficits and the increases in leukocyte venular adherence and tissue infiltration but significantly reduced the magnitude of tissue injury. CONCLUSION: Our findings suggest that mannitol may be of limited value for the prevention of early reperfusion-induced injury after no-flow ischemia in skeletal muscle. By comparison, DMTU was highly efficacious by not only reducing microvascular perfusion deficits but by also reducing leukocyte-endothelial cell interactions and the incidence of cellular injury. (+info)
DNA minor groove recognition by bis-benzimidazole analogues of Hoechst 33258: insights into structure-DNA affinity relationships assessed by fluorescence titration measurements.
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Fluorescence titration measurements have been used to examine the binding interaction of a number of analogues of the bis -benzimidazole DNA minor groove binding agent Hoechst 33258 with the decamer duplex d(GCAAATTTGC)2. The method of continuous variation in ligand concentration (Job plot analysis) reveals a 1:1 binding stoichiometry for all four analogues; binding constants are independent of drug concentration (in the range [ligand] = 0.1-5 microM). The four analogues studied were chosen in order to gain some insight into the relative importance of a number of key structural features for minor groove recognition, namely (i) steric bulk of the N -methylpiperazine ring, (ii) ligand hydrophobicity, (iii) isohelicity with the DNA minor groove and (iv) net ligand charge. This was achieved, first, by replacing the bulky, non-planar N -methylpiperazine ring with a less bulky planar charged imidazole ring permitting binding to a narrower groove, secondly, by linking the N -methylpiperazine ring to the phenyl end of the molecule to give the molecule a more linear, less isohelical conformation and, finally, by introducing a charged imidazole ring in place of the phenolic OH making it dicationic, enabling the contribution of the additional electrostatic interaction and extended conformation to be assessed. Delta G values were measured at 20 degrees C in the range -47.6 to -37.5 kJ mol-1 and at a number of pH values between 5.0 and 7.2. We find a very poor correlation between Delta G values determined by fluorescence titration and effects of ligand binding on DNA melting temperatures, concluding that isothermal titration methods provide the most reliable method of determining binding affinities. Our results indicate that the bulky N -methylpiperazine ring imparts a large favourable binding interaction, despite its apparent requirement for a wider minor groove, which others have suggested arises in a large part from the hydrophobic effect. The binding constant appears to be insensitive to the isohelical arrangement of the constituent rings which in these analogues gives the same register of hydrogen bonding interactions with the floor of the groove. (+info)
Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining.
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The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa. (+info)
Autosomal trisomy 20 (61,XX,+20) in a malformed bovine fetus.
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A 240-day-gestation female bovine fetus with severe anasarca, palatoschisis, cheiloschisis, mild cranioschisis, and a flattened facies was collected at a slaughterhouse, and a fibroblast line was established from the fetal skin. Chromosome preparations were Q-banded, and chromosome counts were taken that indicated the presence of 61 chromosomes in cells of the fetus (the normal diploid number for domestic cattle is 60). Q-band karyotypes were constructed, and Q-band analysis revealed the presence of three copies of chromosome 20. Trisomy 20 (61,XX,+20) was confirmed through the use of two-color fluorescence in situ hybridization of bovine bacterial artificial chromosome clones that were specific to chromosome 20 and the X chromosome. (+info)
Massive parallel analysis of DNA-Hoechst 33258 binding specificity with a generic oligodeoxyribonucleotide microchip.
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A generic oligodeoxyribonucleotide microchip was used to determine the sequence specificity of Hoechst 33258 binding to double-stranded DNA. The generic microchip contained 4096 oxctadeoxynucleo-tides in which all possible 4(6)= 4096 hexadeoxy-nucleotide sequences are flanked on both the 3'- and 5'-ends with equimolar mixtures of four bases. The microchip was manufactured by chemical immobilization of presynthesized 8mers within polyacrylamide gel pads. A selected set of immobilized 8mers was converted to double-stranded form by hybridization with a mixture of fluorescently labeled complementary 8mers. Massive parallel measurements of melting curves were carried out for the majority of 2080 6mer duplexes, in both the absence and presence of the Hoechst dye. The sequence-specific affinity for Hoechst 33258 was calculated as the increase in melting temperature caused by ligand binding. The dye exhibited specificity for A:T but not G:C base pairs. The affinity is low for two A:T base pairs, increases significantly for three, and reaches a plateau for four A:T base pairs. The relative ligand affinity for all trinucleotide and tetranucleotide sequences (A/T)(3)and (A/T)(4)was estimated. The free energy of dye binding to several duplexes was calculated from the equilibrium melting curves of the duplexes formed on the oligonucleotide microchips. This method can be used as a general approach for massive screening of the sequence specificity of DNA-binding compounds. (+info)
Synergistic cytotoxicity and apoptosis by Apo-2 ligand and adriamycin against bladder cancer cells.
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Resistance to conventional anticancer chemotherapeutic agents remains one of the major problems in the treatment of bladder cancer. Hence, new therapeutic modalities are necessary to treat the drug-resistant cancers. Apo-2 ligand (Apo-2L) is member of the tumor necrosis factor ligand family, and it induces apoptosis in cancer cells. Several cytotoxic anticancer drugs, including Adriamycin (ADR), also mediate apoptosis and may share the common intracellular pathways leading to cell death. We reasoned that combination treatment of the drug-resistant cancer cells with Apo-2L and drugs might overcome their resistance. Here, we examined whether bladder cancer cells are sensitive to Apo-2L-mediated cytotoxicity and whether Apo-2L can synergize with ADR in cytotoxicity and apoptosis against bladder cancer cells. Recombinant human soluble Apo-2L (sApo-2L), which carries the extracellular domain of Apo-2L, was used as a ligand. Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. Human T24 bladder cancer line was relatively resistant to sApo-2L. Treatment of T24 line with combination of sApo-2L and ADR resulted in a synergistic cytotoxic effect. Synergy was also achieved in the ADR-resistant T24 line (T24/ADR), two other bladder cancer lines, and three freshly derived human bladder cancer cell samples. In addition, T24 cells were sensitive to treatment with sApo-2L combined with epirubicin or pirarubicin. The synergy achieved in cytotoxicity with sApo-2L and ADR was also achieved in apoptosis. Intracellular accumulation of ADR was not affected by sApo-2L. Incubation of T24 cells with sApo-2L down-regulated the expression of glutathione S-transferase-pi mRNA. This study demonstrates that combination treatment of bladder cancer cells with sApo-2L and ADR overcomes their resistance. The sensitization obtained with established ADR-resistant bladder cancer cells and freshly isolated bladder cancer cells required low subtoxic concentrations of ADR, thus supporting the in vivo potential application of combination of sApo-2L and ADR in the treatment of ADR-resistant bladder cancer. (+info)
DNA cleavage by hydroxy-salicylidene-ethylendiamine-iron complexes.
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Bis(hydroxy)salen.Fe complexes were designed as self-activated chemical nucleases. The presence of a hy-droxyl group on the two salicylidene moieties serve to form a hydroquinone system cooperating with the iron redox system to facilitate spontaneous formation of free radicals. We compared the DNA binding and cleaving properties of the ortho -, meta- and para -(bishydroxy) salen.Fe complexes with that of the corresponding chelate lacking the hydroxyl groups. DNA melting temperature studies indicated that the para complex exhibits the highest affinity for DNA. In addition, this para compound was considerably more potent at cleaving supercoiled plasmid DNA than the regio-isomeric ortho - and meta -hydroxy-salen.Fe complexes, even in the absence of a reducing agent, such as dithiothreitol used to activate the metal complex. The DNA cleaving activity of the para isomer is both time and concentration dependent and the complexed iron atom is absolutely essential for the sequence uniform cleavage of DNA. From a mechanistic point of view, electron spin resonance measurements suggest that DNA contributes positively to the activation of the semi-quinone system and the production of ligand radical species responsible for subsequent strand scission in the absence of a reducing agent. The para -hydroxy-salen.Fe complex has been used for detecting sequence-specific drug-DNA interactions. Specific binding of Hoechst 33258 to AT sequences and chromomycin to GC sequences were shown. The para -bis(hydroxy)salen.Fe derivative complements the tool box of footprinting reagents which can be utilised to produce efficient cleavage of DNA. (+info)