Signaling Defect in the Activation of Caspase-3 and PKCδ in Human TUR Leukemia Cells Is Associated with Resistance to Apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-β- -arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G₀/G₁of the cell cycle and an accumulation of a population in the sub-G₁phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measuredin vitroby enhanced metabolization of a fluorescence substrate andin vivoby cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cδ. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

Zonation of spiders (Araneae) and carabid beetles (Coleoptera: Carabidae) in island salt marshes at the North Sea coast

The specific communities of spiders and carabid beetles of island salt marsh habitats of the East Frisian Island chain at the German North Sea coast were investigated. During the vegetation periods of 1997 and 1998 three pitfall trapping transects were installed on the islands of Borkum and Wangerooge. Within the salt marshes, transects extended from 0 m to 175 m. Elevation gradients varied between 10 cm and 232 cm above MHT (mean high tide). On Borkum, 35 traps were exposed in two transects, on Wangerooge 25 traps were placed in one transect. Three to five elevations above MHT were investigated per transect, each one with five traps. Highest species numbers were recorded in the higher elevated salt marshes. In contrast, highest activity values were noticed in the medium elevated salt marshes. Within both groups, spiders and carabids, four communities were distinguished by indirect gradient analysis. Indicator species were assigned to the different communities that were mainly assorted to different elevations of the salt marshes. Thus, the communities of both taxa corresponded well to the vegetational formations. The importance of sea level rise for structuring the communities of salt marsh arthropods is discussed. Overall, still great uncertainties exist on how arthropod communities and salt marshes themselves will develop.     

Models for the Generation and Interpretation of Gradients

Source regions for morphogen gradients—organizing regions—can be generated if a local self-enhancing reaction is coupled with a long-ranging reaction that acts antagonistically. Resulting gradients can be translated into patterns of stable gene activities using genes whose products have a positive feedback on the activation on themselves. If several autoregulatory genes compete with each other for activity, cells make an unequivocal choice. Although the signal consists of a smoothly graded distribution, the all-or-nothing response of the cells leads to regions of differently determined cells that are delimited by sharp borders. In some systems, it is not the absolute but the relative level of a gradient that matters. The sequence of head, tentacles, and foot formation in hydra is controlled by a head activation gradient and is an example of this widely used but conceptually rather neglected mode. For subpatterns such as legs and wings, different “compartments” cooperate to produce new signaling substances. Here, morphogen production is restricted to the common borders or where they intersect. The model accounts for the formation of substructures in pairs at the correct positions within the embryo and for the correct orientation and handedness with respect to the main body axes.Many observations in developmental biology can be accounted for by the assumption that graded distributions of substances control cell differentiation in a concentration-dependent manner. Earlier objections against this concept came from two sides. First, the assumption of gradients shifts only the problem as long as no explanation is given of how the gradients are generated in the first place. Second, cells must be extremely sensitive in measuring the absolute concentrations. Only minute concentration differences are expected to exist between adjacent cells. Nevertheless, these must be sufficient to accomplish a reliable and unequivocal selection between alternative pathways.Long before the molecular approach became feasible, we proposed models showing that these features can be realized by molecularly reasonable interactions. Meanwhile, these models found strong support by molecular and genetic observations.     

Intracellular dissemination of peroxidative stress. Internalization, transport, and lethal targeting of a cholesterol hydroperoxide species by sterol carrier protein-2-overexpressing hepatoma cells

Sterol carrier protein-2 (SCP-2) plays a crucial role in the trafficking and metabolism of cholesterol and other lipids in mammalian cells. Lipid hydroperoxides generated under oxidative stress conditions are relatively long-lived intermediates that damage cell membranes and play an important role in redox signaling. We hypothesized that SCP-2-facilitated translocation of lipid hydroperoxides in oxidatively stressed cells might enhance cytolethality if highly sensitive sites are targeted and detoxification capacity is insufficient. We tested this using a clone (SC2A) of rat hepatoma cells that overexpress mature immunodetectable SCP-2. When challenged with liposomal cholesterol-7alpha-hydroperoxide (7alpha-OOH), SC2A cells were found to be much more sensitive to viability loss than vector control (VC) counterparts. Correspondingly, SC2A cells imported [14C]7alpha-OOH more rapidly. The clones were equally sensitive to tert-butyl hydroperoxide, suggesting that the 7alpha-OOH effect was SCP-2-specific. Fluorescence intensity of the probes 2',7'-dichlorofluorescein and C11-BODIPY increased more rapidly in SC2A than VC cells after 7alpha-OOH exposure, consistent with more rapid internalization and oxidative turnover in the former. [14C]7alpha-OOH radioactivity accumulated much faster in SC2A mitochondria than in VC, whereas other subcellular fractions showed little rate difference. In keeping with this, 7alpha-OOH-stressed SC2A cells exhibited a faster loss of mitochondrial membrane potential and development of apoptosis. This is the first reported evidence that peroxidative stress damage can be selectively targeted and exacerbated by an intracellular lipid transfer protein.     

Molecular Characterization of Cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis>) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important commodity in Jamaica. Knowledge of the genetic diversity of Jamaican cacao germplasm is essential for their conservation and management. In spite of cacao’s economic importance in Jamaica, the crop is under studied, therefore limiting sound decisions toward improving productivity. Assessment of germplasm and on-farm genetic diversity is required to assist selecting superior genotypes to propagate and distribute across the island, as well as to use them as parental clones in breeding programs. Using 94 single nucleotide polymorphism (SNP) markers, 140 Jamaican cacao samples from two germplasm collections and a farmer’s estate along with 150 reference samples were analyzed. The principal coordinate analysis demonstrated that the majority of the Jamaican cacao selections were hybrids derived from five original germplasm groups, including Criollo, Amelonado and three Upper Amazon Forastero groups. Among the Upper Amazon groups, the Bayesian clustering analysis revealed that the Parinari (PA) ancestral lineage contributed the most (29.9%) to the Jamaican cacao germplasm. The germplasm collections showed greater diversity in terms of ancestral contributions compared to the farmer’s estate. However, the genetic differentiation between the three collecting sites was small (Fst?=?0.036), indicating that samples collected from the three sites were derived from a common pool of germplasm. The current study supports the historical records and clarified the ancestry of Jamaican cacao. Although the majority of the cacao genetic groups were observed in the Jamaican cacao collections, several diversity gaps were found in both germplasm collections and in the farmer’s estate, especially germplasm with disease resistance to cacao frosty pod rot that was recently found in Jamaica.     

The Tsn1-ToxA interaction in the wheat-Stagonospora nodorum pathosystem parallels that of the wheat-tan spot system.

The wheat tan spot fungus (Pyrenophora tritici-repentis) produces a well-characterized host-selective toxin (HST) known as Ptr ToxA, which induces necrosis in genotypes that harbor the Tsn1 gene on chromosome 5B. In previous work, we showed that the Stagonospora nodorum isolate Sn2000 produces at least 2 HSTs (SnTox1 and SnToxA). Sensitivity to SnTox1 is governed by the Snn1 gene on chromosome 1B in wheat. SnToxA is encoded by a gene with a high degree of similarity to the Ptr ToxA gene. Here, we evaluate toxin sensitivity and resistance to S. nodorum blotch (SNB) caused by Sn2000 in a recombinant inbred population that does not segregate for Snn1. Sensitivity to the Sn2000 toxin preparation cosegregated with sensitivity to Ptr ToxA at the Tsn1 locus. Tsn1-disrupted mutants were insensitive to both Ptr ToxA and SnToxA, suggesting that the 2 toxins are functionally similar, because they recognize the same locus in the host to induce necrosis. The locus harboring the tsn1 allele underlies a major quantitative trait locus (QTL) for resistance to SNB caused by Sn2000, and explains 62% of the phenotypic variation, indicating that the toxin is an important virulence factor for this fungus. The Tsn1 locus and several minor QTLs together explained 77% of the phenotypic variation. Therefore, the Tsn1-ToxA interaction in the wheat-S. nodorum pathosystem parallels that of the wheat-tan spot system, and the wheat Tsn1 gene serves as a major determinant for susceptibility to both SNB and tan spot.