Accumulation of short telomeres in human fibroblasts prior to replicative senescence

The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1-2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5-1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence.     

Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions

Galleria mellonella larvae cultured axenically were treated with axenic dauer juveniles of Heterorhabditis bacteriophora and Steinernema carpocapsae. After 3 days S. carpocapsae had killed all insects, with 9.4 +/- 4.3 nematodes per larva. H. bacteriophora were unable to kill G. mellonella, although 13.3 +/- 6.4 nematodes per Galleria were found in the hemocoel. Invading nematodes of both strains recovered from the dauer stage. H. bacteriophora developed into hermaphrodites with eggs and J1 in the uterus and in the hemolymph of the living insects. Development beyond the J1 stage was not recorded. An injection of supernatants from different Photorhabdus luminescens cultures killed the insects but could not provide nutrients to support a further development. Only the injection of bacterial cells supported production of dauers in the axenic insects. Axenic S. carpocapsae developed to adults and produced offspring. After 3 weeks an average of 5275 nematodes per larva were counted, of which 6.7% were dauer juveniles, 39.2% other juvenile stages, 11.9% males, and 42.2% females. Compared to in vivo reproduction in the presence of the symbiotic bacterium Xenorhabdus nematophilus the dauer juvenile yields were low. Even after 5 weeks the percentage of dauer juveniles did not surpass 10%.     

Biodegradation of Reactive Blue 59 by isolated bacterial consortium PMB11

Morphologically different, three bacterial strains, capable of decolorizing Reactive Blue 59 were isolated from dye effluent contaminated soil sample, collected from Ichalkaranji, India. The individual bacterial strains viz. Bacillus odysseyi SUK3, Morganella morganii SUK5 and Proteus sp. SUK7 decolorized Reactive Blue 59 (50 mg l(-1)) completely within 60, 30, 24 h, respectively, while the bacterial consortium PMB11 of these strains required 3 h for the complete decolorization. The decolorization was confirmed by UV-Vis spectroscopy. Further, the biodegradation of Reactive Blue 59 in to different metabolites was confirmed by High performance liquid chromatography and Fourier transform infrared spectroscopy analysis. Significant increase in the activity of aminopyrine N-demethylase (AND) in the individual as well consortium cells, obtained after decolorization showed involvement of AND in the decolorization process. Phytotoxicity studies, revealed the nontoxic nature of the degraded metabolites of Reactive Blue 59 indicating effectiveness of bacterial consortium PMB11 for the treatment of textile effluent containing Reactive Blue 59.     

Growth kinetics of Escherichia coli with galactose and several other sugars in carbon-limited chemostat culture

Kinetic models for microbial growth describe the specific growth rate (mu) as a function of the concentration of the growth-limiting nutrient (s) and a set of parameters. A typical example is the model proposed by Monod, where mu is related to s using substrate affinity (Ks) and the maximum specific growth rate (mu max). The preferred method to determine such parameters is to grow microorganisms in continuous culture and to measure the concentration of the growth-limiting substrate as a function of the dilution rate. However, owing to the lack of analytical methods to quantify sugars in the microgram per litre range, it has not been possible to investigate the growth kinetics of Escherichia coli in chemostat culture. Using an HPLC method able to determine steady-state concentrations of reducing sugars, we previously have shown that the Monod model adequately describes glucose-limited growth of E. coli ML30. This has not been confirmed for any other sugar. Therefore, we carried out a similar study with galactose and found steady-state concentrations between 18 and 840 micrograms.L-1 for dilution rates between 0.2 and 0.8.h-1, respectively. With these data the parameters of several models giving the specific growth rate as a function of the substrate concentration were estimated by nonlinear parameter estimation, and subsequently, the models were evaluated statistically. From all equations tested, the Monod model described the data best. The parameters for galactose utilisation were mu max = 0.75.h-1 and Ks = 67 micrograms.L-1. The results indicated that accurate Ks values can be estimated from a limited set of steady-state data when employing mu max measured during balanced growth in batch culture. This simplified procedure was applied for maltose, ribose, and fructose. For growth of E. coli with these sugars, mu max and Ks were for maltose 0.87.h-1, 100 micrograms.L-1; for ribose 0.57.h-1, 132 micrograms.L-1, and for fructose 0.70.h-1, 125 micrograms.L-1.     

Physical map of the chromosome of the apple proliferation phytoplasma

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.     

Reduced interleukin-1 responsiveness in immune system and central nervous system of inbred polydipsic (STR/N) mice

Inbred polydipsic mice (STR/N strain) have primary polydipsia. The previous studies found abnormalities in the central nervous system (CNS), especially in the hypothalamus and circumventricular organ. As a part of pursuing to find the cause of the polydipsia, we investigated immunological characteristics of STR/N mice, using the ICR strain of mice as control. Their thymic subset cells showed that CD4+CD8+ double positive cells were increased, CD4+ single positive cells were decreased and CD5 expression was deficient, compared to ICR mice. T cell proliferative response and interleukin (IL)-2 production caused by IL-1beta stimulation were reduced in STR/N mice than those in the ICR mice. In in vivo studies the degree of thymic atrophy and the increases in serum level of ACTH and corticosterone induced by intraperitoneal IL-1beta injection were much less in STR/N mice than those in controls. Furthermore, adipsic response also induced by IL-1beta injection was greatly reduced compared to their control mice. All these results suggest that the responsiveness to IL-1 is impaired both in the immune system and the CNS of STR/N mice.     

Chilling tolerance of Central European maize lines and their factorial crosses

BACKGROUND AND AIMS: Chilling-stress tolerance is a prerequisite for maize production under cool climatic conditions. The main goal of this study was to evaluate the Central European dent and flint pools for chilling tolerance during heterotrophic and early autotrophic growth in field trials and growth chamber experiments. METHODS: Five European flint and five dent inbreds and their 25 factorial crosses were evaluated in six natural environments, where chilling occurred, for chlorophyll concentration and plant height at the three-leaf stage, and plant height and fresh weight at the seven-leaf stage. In growth chambers, leaf 3 growth was analysed under cold and control conditions. KEY RESULTS: Comparing the field and growth chamber data, the strongest association was found between leaf elongation rate during cold nights and plant height at the three-leaf stage, with a weaker association with the seven-leaf stage. In the field, moderate correlations were observed between plant height at the three-leaf stage, and plant height and fresh weight at the seven-leaf stage, respectively. Furthermore, mid-parent and hybrid performance were only moderately correlated. CONCLUSIONS: The results suggest that heterotrophic and early autotrophic growth stages are controlled by different genetic factors or that maternal effects play a role. In addition, the findings showed that mid-parent performance is a poor predictor of hybrid performance. Consequently, test cross performance should be the target in quantitiative trait locus (QTL) mapping studies with the final goal of establishing marker-assisted breeding programmes for chilling-tolerant hybrids.     

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 ? resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.