Glycinebetaine enhances and stabilizes the evolution of oxygen and the synthesis of ATP by cyanobacterial thylakoid membranes.

Glycinebetaine (betaine), an osmoregulant in halophilic plants, stabilized the evolution of oxygen and the synthesis of ATP by thylakoid membranes from the cyanobacterium Synechocystis PCC6803 when it was present during the preparation and incubation of the thylakoid membranes. Moreover, betaine enhanced the evolution of oxygen and the synthesis of ATP when present during assays. When betaine at 1.0 M was present during the preparation of thylakoid membranes and during the measurement of activity, the rate of evolution of oxygen was equivalent to that of intact cells.     

Direct measurement of static chest wall compliance in animal and human neonates

The measurement of pulmonary mechanics has been developed extensively for adults, and these techniques have been applied directly to neonates and infants. However, the compliant chest wall of the infant frequently predisposes to chest wall distortion, especially when there is a low dynamic lung compliance (CL,dyn). We describe a technique of directly measuring the static chest wall compliance (Cw,st), developed initially in the newborn lamb and subsequently applied to the premature neonate with chest wall distortion. The mean CL,dyn in seven intubated newborn lambs in normoxia was 2.45 +/- 0.41 ml.cmH2O-1.kg-1, whereas Cw,st was 11.81 +/- 0.25 ml.cmH2O-1.kg-1. These values did not change significantly in seven animals breathing through a tight-fitting face mask or with hypercapnia-induced tachypnea. For the eight premature infants the mean CL,dyn was 1.35 +/- 0.36 ml.cmH2O-1.kg-1, whereas the mean Cw,st was 3.16 +/- 1.01 ml.cmH2O-1.kg-1. This study shows that, under relaxed conditions when measurements of static compliance are performed, the chest wall is more compliant than the lung. The measurement of Cw,st may thus be used to determine the contribution of the respiratory musculature in stabilizing the chest wall.     

Genetic diversity and population structure of the serpentine endemic Ni hyperaccumulator Alyssum lesbiacum

Alyssum lesbiacum is a well-established Ni hyperaccumulator, endemic to serpentine soils of Lesbos Island (Greece). A total of 95 individuals were collected from 17 plots encompassing the only four large populations of this species. ISSR (Inter-simple sequence repeat) markers were used to assess genetic diversity and population structure of A. lesbiacum to provide initial guidance for the development of successful management and conservation measures. A total of ten primers produced 82 bands, 96.34 % being polymorphic. The largest part of total diversity was found within populations, rather than among them. AMOVA analysis partitioned the largest part of diversity within plots (66 %), while 15 % contained among plots within populations and 19 % among populations. Principal coordinates analysis along with dendrogram based on genetic distances among populations showed a high degree of genetic differentiation of the isolated population Loutra. At a smaller scale, distance was not the most significant factor influencing the patterns of genetic diversity, but topography, ecosystem types and connectivity through streams. According to our results, conservation efforts should be organized at the level of watersheds and ecosystem types, considering them as management units. For ex situ conservation and restoration, seed samplings should be representative of the different habitats and watersheds as well as the patches of large and small populations while keeping the east population of Loutra separate from the other three central populations, to avoid any loss of genetic diversity and to preserve the character of the local adapted populations.     

Differential regulation of stimulated glucose transport by free fatty acids and PPARα or -δ agonists in cardiac myocytes

Stimulation of glucose transport in response to insulin or metabolic stress is an important determinant of cardiac myocyte function and survival, particularly during ischemia-reperfusion episodes. The impact of dyslipidemia and its consequence PPAR activation on stimulated glucose transport in cardiac myocytes remains unknown. Isolated adult rat cardiac myocytes were chronically exposed to free fatty acids (FFA) or PPAR agonists. Insulin- (ISGT) and oligomycin-stimulated glucose transport (OSGT) and related cell signaling were analyzed. Exposure of cardiac myocytes to FFA reduced both ISGT and OSGT. Exposure to either PPARα or PPARδ agonists, but not to a PPARγ agonist, reduced ISGT but not OSGT and increased fatty acid oxidation (FAO). The reduction in ISGT was associated with impaired insulin signaling and, in the case of PPAR stimulation, overexpression of SOCS-3, a protein known to hinder proximal insulin signaling. In contrast, the reduction of OSGT could not be explained by a reduced activity of the cellular energy-sensing system, as assessed from the maintained phosphorylation state of AMPK. Inhibition of FAO at the level of mitochondrial acylcarnitine uptake restored OSGT but not ISGT. Seemingly paradoxically, further stimulation of FAO with PPARα or PPARδ agonists also restored OSGT but not ISGT. Together, these results suggest that inhibition of OSGT occurs downstream of energy gauging and is caused by some intermediate(s) of fatty acid oxidation, which does not appear to be acylcarnitines. The results indicate that the mechanisms underlying FFA-mediated inhibition of ISGT and OSGT differ remarkably.     

Identification of a secondary zinc-binding site in staphylococcal enterotoxin C2. Implications for superantigen recognition

The previously determined crystal structure of the superantigen staphylococcal enterotoxin C2 (SEC2) showed binding of a single zinc ion located between the N- and C-terminal domains. Here we present the crystal structure of SEC2 determined to 2.0 A resolution in the presence of additional zinc. The structure revealed the presence of a secondary zinc-binding site close to the major histocompatibility complex (MHC)-binding site of the toxin and some 28 A away from the primary zinc-binding site of the toxin found in previous studies. T cell stimulation assays showed that varying the concentration of zinc ions present affected the activity of the toxin and we observed that high zinc concentrations considerably inhibited T cell responses. This indicates that SEC2 may have multiple modes of interaction with the immune system that are dependent on serum zinc levels. The potential role of the secondary zinc-binding site and that of the primary one in the formation of the TCR.SEC2.MHC complex are considered, and the possibility that zinc may regulate the activity of SEC2 as a toxin facilitating different T cell responses is discussed.     

HU histone-like DNA-binding protein from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: structural and evolutionary analyses

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus–Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.     

Spectral characteristic of fluorescence induction in a model cyanobacterium, Synechococcus sp. (PCC 7942)

We present here three-dimensional time-wavelength-intensity displays of changes in variable fluorescence, during the O(JI)PSMT transient, observed in cyanobacterium at room temperature. We were able to measure contributions of individual chromophores to fluorescence spectra at various times of fluorescence induction (FI). The method was applied to a freshwater cyanobacterium, Synechococcus sp. (PCC 7942). Analysis of our experimental results provides the following new conclusions: (i) the main chlorophyll (Chl) a emission band at ∼ 685 nm that originates in Photosystem (PS) II exhibits typical fast (OPS) and slow (SMT) FI kinetics with both orange (622 nm) and blue (464 nm) excitation. (ii) Similar kinetics are exhibited for its far-red emission satellite band centered at ∼ 745 nm, where the PS II contribution predominates. (iii) A significant OPS-SMT-type kinetics of C-phycocyanin emission at ∼ 650 nm are observed with the blue light excitation, but not with orange light excitation where the signal rose only slightly to a maximum. The induction of F650 was not caused by an admixture of the F685 fluorescence and thus our data show light-inducible and dark-reversible changes of phycobilin fluorescence in vivo. We discuss possible interpretations of this new observation.     

Trace constituents in the essential oil of Thymus capitatus

The essential oil of Thymus capitatus was investigated by glass capillary gas chromatography in combination with mass spectrometry. In the analysis, 22 hitherto unreported trace constituents were isolated, of which 20 were identified.     

Diverse supernumerary structures develop after inverting the anteroposterior limb axis of the anuran

Contralateral limb bud graftings were performed on tadpoles of the anuran Bufo bufo. The anteroposterior axis was inverted while the larvae were at stage IV or V (e.g., between 22-30 days after fertilization). Eighty-four tadpoles were operated on, 10 of which were used as controls. At anterior or posterior location 104 supernumerary structures developed in toto. They were collected and whole-mount examined after being stained with Alcian blue. They were further prepared for serial sectioning, mounting, and staining with hematoxylin and eosin. The majority of these supernumerary structures were found to be normal limbs of the stump handedness in agreement with all models and experiments on the urodeles axolotl and newt. However, some of the structures were clearly abnormal: double symmetric or of mixed handedness. This result is consistent with a prediction of a hierarchical polar coordinate model. The fact that no such structures have been found in the experiments on the urodeles may be due to the expected low probability for their appearance and the fact that only few such limbs have been sectioned and analyzed as yet.     

A morphogen gradient model for pattern regulation. II. Time description of global morphogen formation and field compartmentalization

In a model for pattern regulation, use was made of local and global morphogens S and Sigma. Sigma is produced from the S-degradation and it is decomposed by first order kinetics while it diffuses along the field. We solve exactly the partial differential equation for the distribution of Sigma in one spatial dimension when its source S is monotonie (for simplicity, linear or generally a power function). Assuming that S and Sigma react reversibly with an allosteric protein P according to a sequential scheme, we derive the evolution in time of the field separation into compartments. At equilibrium the relative extent of each compartment is constant (for variable field size) and so pattern regulation is achieved.