Responses of Azospirillum brasilense to Nitrogen Deficiency and to Wheat Lectin: A Diffuse Reflectance Infrared Fourier Transform (DRIFT) Spectroscopic Study

For the rhizobacterium Azospirillum brasilense, the optimal nutritional range of C:N ratios corresponds to the presence of malate (ca. 3 to 5 g l⁻¹ of its sodium salt) and ammonium (ca. 0.5 to 3 g l⁻¹ of NH₄Cl) as preferred carbon and nitrogen sources, respectively. This microaerophilic aerotactic bacterium is known to have a narrow optimal oxygen concentration range of ca. 3 to 5 μM, which is 1.2% to 2% of oxygen solubility in air-saturated water under normal conditions. In this work, the effects of stress conditions (bound-nitrogen deficiency related to a high C:N ratio in the medium; excess of oxygen) on aerobically grown A. brasilense Sp245, a native wheat-associated endophyte, were investigated in the absence and presence of wheat germ agglutinin (WGA, plant stress protein and a molecular host-plant signal for the bacterium) using FTIR spectroscopy of whole cells in the diffuse reflectance mode (DRIFT). The nutritional stress resulted in the appearance of prominent spectroscopic signs of poly-3-hydroxybutyrate (PHB) accumulation in the bacterial cells; in addition, splitting of the amide I band related to bacterial cellular proteins indicated some stress-induced alterations in their secondary structure components. Similar structural changes were observed in the presence of nanomolar WGA both in stressed A. brasilense cells and under normal nutritional conditions. Comparative analysis of the data obtained and the relevant literature data indicated that the stress conditions applied (which resulted in the accumulation of PHB involved in stress tolerance) and/or the presence of nanomolar concentrations of WGA induced synthesis of bacterial cell-surface (glyco)proteins rich in β-structures, that could be represented by hemagglutinin and/or porin.     

Mydj2 as a potent partner of hsc70 in mammalian cells.

Dj2 is a member of the DnaJ family of proteins, which regulate the chaperoning function of the hsp70s. We isolated a monkey cDNA dj2 clone corresponding to the large mRNA species encoded by the gene. This mRNA differs from the small mRNA produced by the same gene in that it contains a long 3' untranslated region. Both messages were found to be equally stable and to produce the same protein, which is susceptible to farnesylation. Studies in mouse tissues and various cell lines revealed that these messages and their products are differentially expressed. Surprisingly, we found that only the nonfarnesylated form of dj2 is capable of translocating to the cell nucleus, especially after heat shock. Finally, based on protein interaction studies, our results indicate that dj2 is a specific partner for hsc70 and not for hsp70.     

Population Genomics of Wall Lizards Reflects the Dynamic History of the Mediterranean Basin

The Mediterranean Basin has experienced extensive change in geology and climate over the past six million years. Yet, the relative importance of key geological events for the distribution and genetic structure of the Mediterranean fauna remains poorly understood. Here, we use population genomic and phylogenomic analyses to establish the evolutionary history and genetic structure of common wall lizards (Podarcis muralis). This species is particularly informative because, in contrast to other Mediterranean lizards, it is widespread across the Iberian, Italian, and Balkan Peninsulas, and in extra-Mediterranean regions. We found strong support for six major lineages within P. muralis, which were largely discordant with the phylogenetic relationship of mitochondrial DNA. The most recent common ancestor of extant P. muralis was likely distributed in the Italian Peninsula, and experienced an “Out-of-Italy” expansion following the Messinian salinity crisis (∼5 Mya), resulting in the differentiation into the extant lineages on the Iberian, Italian, and Balkan Peninsulas. Introgression analysis revealed that both inter- and intraspecific gene flows have been pervasive throughout the evolutionary history of P. muralis. For example, the Southern Italy lineage has a hybrid origin, formed through admixture between the Central Italy lineage and an ancient lineage that was the sister to all other P. muralis. More recent genetic differentiation is associated with the onset of the Quaternary glaciations, which influenced population dynamics and genetic diversity of contemporary lineages. These results demonstrate the pervasive role of Mediterranean geology and climate for the evolutionary history and population genetic structure of extant species.     

Hydrogen peroxide and catalase are inversely related in adult patients undergoing cardiopulmonary bypass: implications for antioxidant protection

Adult patients undergoing cardiopulmonary bypass (CPB) surgery are subjected to increased oxidative stress and show a spectrum of lung injury. Increased levels of hydrogen peroxide (H2O2) are often seen during episodes of oxidative stress, such as the use of high FiO2s, and this molecule plays a key role in the formation of highly damaging oxidants such as the hydroxyl radical. Oxidative damage to plasma proteins was assessed by measuring free thiol groups, and antioxidant protection against H2O2 by measuring catalase activity. CPB patients (n = 39) receiving either 100% or 50% oxygen at the end of bypass were studied by measuring levels of H2O2 in breath condensate and levels of catalase in their plasma, and comparing these to pre-bypass levels. Post-bypass, all CPB patients exhaled significantly lower levels of H2O2 (P < 0.0001) at a time when they had significantly increased activity (0.809 +/- 0.11 versus 1.688 +/- 0.18 U/mg protein) of catalase in their plasma. There were no significant differences in these parameters between the 100% and 50% oxygen groups. At a time when oxidative stress is greatest, there appears to be a corresponding plasma increase in the antioxidant catalase. Whether this change is fortuitous or a response to oxidative stress is at present under consideration.     

Seed and cone diversity and seed germination of Pinus pinea in Strofylia Site of the Natura 2000 Network

Variation in cone size, seed number per cone, seed potential, seed efficiency, seed morphology and seed germination behavior of Pinus pinea and its relation to stand conditions was analyzed. Data were collected from P. pinea forest in Strofylia, southern Greece, a forest that belongs to the Natura 2000 European network and the RAMSAR convention and is characterized by the absence of regeneration for many decades. The pine stands found in the area were distinguished into five categories according to a previous study and our observations, regarding stand age, canopy cover and the degree of stress by human pressure. The categories are: (I) young artificial, (II) closed-mature, (III) open-mature, (IV) over-mature stands and (V) highly degraded stands. Cones were collected from all stand types and their morphological characteristics as well as their seed production were measured. Seeds were extracted from the collected cones, measured and their germination behavior was tested. The findings showed that the over-mature and the high degraded stands and to a lesser extent, the closed-mature stands, produced significantly smaller cones with a lower seed potential, a lower number of filled seeds per cone, a greater number of not fully developed seeds and reduced seed morphological characteristics than the young and open-mature stands. However, the seed germination behavior of fully developed seeds was only slightly affected by the stand type.     

Positive contrast in the detection of magnetically labeled cells by MRI--in vitro experiments]

PURPOSE: Magnetically labeled cells (MLC) cause local field inhomogenities within the single voxels as well as on a macroscopic scale. The related Larmor frequency shift near MLC was exploited to obtain bright visualization applying spectral selective saturation (SSS). METHODS: SK-Mel28 cells were labeled with the superparamagnetic iron oxide contrast agent SHU 555A. Low cell concentrations (0, 5, 20, 30, 50, 75, and 150 MLC/microl) and high cell concentrations (10 x 10(3), 30 x 10(3), 60 x 10(3), and 100 x 10(3) MLC/10 microl) were examined at 3 Tesla. Shimming and frequency adjustment to spectrometer reference frequency v0 was performed with the built in routine of the scanner. A 2D spin echo sequence with broadband excitation and refocusing pulses was used (BWex = 1.000 Hz). Prior to each TR, a non-selective saturation sinc pulse centred at v0 was applied. Bandwidth (BWsat) of this pulse was varied from 100 Hz to 800 Hz in logarithmic steps. RESULTS: Without SSS the highest value of Crel (i.e. relative MR contrast between labeled to unlabeled samples) was found for 150 MLC/microl and was given by 10%. Applying SSS led to positive contrast of the complete labeled volumes and to remarkable improvements in Crel. With increasing cell concentrations Crel raised to maximum, that was given by 52% (BWsat=100 Hz) and 28% (BWsat = 200 Hz) found for 75 MLC/microl. For 150 MLC/microl Crel decreased. A contrast clarification could also be detected near cell aggregations despite saturation. CONCLUSION: Using SSS positive contrast can be achieved for voxels containing MLC and voxels close to cell clusters. Under in vitro conditions positive contrast improved the sensitivity to detect MLC as compared to negative contrast imaging techniques. It seems reasonable, that positive contrast approaches can be applied in vivo as the underlying physical mechanism are comparable.     

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.