Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

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.     

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.     

Mechanism of cis-trans isomerization of unsaturated fatty acids in Pseudomonas putida

We studied the pattern of the cis-trans isomerization of unsaturated fatty acids in cells of Pseudomonas putida S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond. Direct evidence that isomerization does not include a transient saturation of the double bond was obtained. In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome c type.     

Complex pattern formation by a self-destabilization of established patterns: chemotactic orientation and phyllotaxis as examples

Stable patterns can be generated by molecular interactions involving local self-enhancement and long-range inhibition. In contrast, highly dynamic patterns result if the maxima, generated in this way, become destabilized by a second antagonistic reaction. The latter must act local and must be long-lasting. Maxima either disappear and reappear at displaced positions or they move over the field as travelling waves. The wave can have unusual properties in that they can penetrate each other without annihilation. The resulting pattern corresponds to those observed in diverse biological systems. In the chemotactic orientation of cells, the temporary signals allow the localized extensions of protrusions under control of minute external asymmetries imposed by the chemoattractant. In phyllotaxis, these signals lead to successive leaf initiation, whereby the longer-lasting extinguishing reaction can cause a displacement of the subsequent leaf initiation site by the typical 137.5 degrees, the golden angle. On seashells, this patterns leads either to oblique lines that can cross each other or to oblique rows of dots. For some of the models animated simulations are available at http://www.eb.tuebingen.mpg.de/abt.4/meinhardt/theory.html.     

Genome organization of the linear Pichia etchellsii plasmid pPE1A: evidence for expression of an extracellular chitin-binding protein homologous to the alpha-subunit of the Kluyveromyces lactis killer toxin

Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) is a non-killer yeast harbouring two cryptic linear cytoplasmic DNA-elements, pPE1A (6.7 kb) and pPE1B (12.8 kb). Cloning and complete sequencing of pPE1A revealed a 6749-bp element with a remarkably high A+T content of 77.6%. The termini of pPE1A were found to consist of inversely orientated identical nucleotide repetitions of 178bp, to which proteins are linked at the 5'-ends. It is only the second small, non-autonomous cytoplasmic yeast linear plasmid for which the complete nucleotide sequence is known. Five open reading frames (ORFs) were identified preceded by upstream conserved sequence motifs (UCS) characteristic for cytoplasmic promoters and perfectly matching the UCS consensus (ATNTGA). As none of the putative genes encodes a DNA-polymerase, pPE1A is the first yeast linear plasmid known that does not possess its own element-specific replication machinery. No function could be attributed to ORF1, 3, 4, and 5; the predicted ORF2 gene product is similar to chitin-binding proteins and chitinases, highest homologies were found to the precursor of the alpha- and beta-subunits of the secreted Kluyveromyces lactis zymocin. Consistently, the Orf2p could be isolated from the culture fluid by chitin-Sepharose affinity chromatography and characterized by immuno-probing with an antibody specific for the K. lactis killer toxin alpha-subunit. Production of the protein was found to be plasmid-dependent. The sequence of pPE1A has been submitted to the EMBL data library, Accession No. AJ409097.     

Necrosis Is the Dominant Cell Death Pathway in Uropathogenic Escherichia coli Elicited Epididymo-Orchitis and Is Responsible for Damage of Rat Testis

Male infertility is a frequent medical condition, compromising approximately one in twenty men, with infections of the reproductive tract constituting a major etiological factor. Bacterial epididymo-orchitis results in acute inflammation most often caused by ascending canalicular infections from the urethra via the continuous male excurrent ductal system. Uropathogenic Escherichia coli (UPEC) represent a relevant pathogen in urogenital tract infections. To explore how bacteria can cause damage and cell loss and thus impair fertility, an in vivo epididymo-orchitis model was employed in rats by injecting UPEC strain CFT073 into the vas deference in close proximity to the epididymis. Seven days post infection bacteria were found predominantly in the testicular interstitial space. UPEC infection resulted in severe impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in sperm numbers and a significant increase in TUNEL (+) cells. Activation of caspase-8 (extrinsic apoptotic pathway), caspase-3/−6 (intrinsic apoptotic pathway), caspase-1 (pyroptosis pathway) and the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic pathway, were not observed in infected testis. Notably, electron microscopical examination revealed degenerative features of Sertoli cells (SC) in UPEC infected testis. Furthermore, the passive release of high mobility group protein B1 (HMGB1), as an indication of necrosis, was observed in vivo in infected testis. Thus, necrosis appears to be the dominant cell death pathway in UPEC infected testis. Substantial necrotic changes seen in Sertoli cells will contribute to impaired spermatogenesis by loss of function in supporting the dependent germ cells.