Physical forces may cause Hox gene collinearity in the primary and secondary axes of the developing vertebrates

The features of spatial and temporal Hox gene collinearity along the anteroposterior and secondary axes of vertebrate development have been extensively studied. However, the understanding of these features remains problematic. Some genetic engineering experiments were performed and the consequent modifications of the Hoxd gene expressions in the vertebrate limb and trunk were presented. A two-phases model was proposed to describe the above results but still many data cannot be explained. In the present work a different mechanism is put forward in order to deal with the above experiments. This alternative mechanism (coined biophysical model), is based on the hypothesis that physical forces decondense and 'loop out' the chromatin fiber causing the observed Hox gene collinearity phenomena at the early stages of axonal development. The two models are compared in detail. The biophysical model adequately explains the data even in cases where the results are characterized as unexpected. Furthermore, the biophysical model predicts that the Hox gene expressions are entangled in space and time and this coupling is compatible with the data of the early developmental stages. Additional experiments are proposed for a direct test of this model.     

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.     

Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.     

A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC7942

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light‐acclimated and of dark‐acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki‐Zorba. 1997. Biochim. Biophys. Acta 1335: 1‐4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na⁺ and K⁺ salts caused rapid (t_1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCl, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCl and water.     

Molecular recognition of human angiogenin by placental ribonuclease inhibitor--an X-ray crystallographic study at 2.0 A resolution.

Human placental RNase inhibitor (hRI), a leucine-rich repeat protein, binds the blood vessel-inducing protein human angiogenin (Ang) with extraordinary affinity (Ki <1 fM). Here we report a 2.0 A resolution crystal structure for the hRI-Ang complex that, together with extensive mutagenesis data from earlier studies, reveals the molecular features of this tight interaction. The hRI-Ang binding interface is large and encompasses 26 residues from hRI and 24 from Ang, recruited from multiple domains of both proteins. However, a substantial fraction of the energetically important contacts involve only a single region of each: the C-terminal segment 434-460 of hRI and the ribonucleolytic active centre of Ang, most notably the catalytic residue Lys40. Although the overall docking of Ang resembles that observed for RNase A in the crystal structure of its complex with the porcine RNase inhibitor, the vast majority of the interactions in the two complexes are distinctive, indicating that the broad specificity of the inhibitor for pancreatic RNase superfamily proteins is based largely on its capacity to recognize features unique to each of them. The implications of these findings for the development of small, hRI-based inhibitors of Ang for therapeutic use are discussed.     

High-Frequency Stimulation of the Subthalamic Nucleus Counteracts Cortical Expression of Major Histocompatibility Complex Genes in a Rat Model of Parkinson’s Disease

High-frequency stimulation of the subthalamic nucleus (STN-HFS) is widely used as therapeutic intervention in patients suffering from advanced Parkinson’s disease. STN-HFS exerts a powerful modulatory effect on cortical motor control by orthodromic modulation of basal ganglia outflow and via antidromic activation of corticofugal fibers. However, STN-HFS-induced changes of the sensorimotor cortex are hitherto unexplored. To address this question at a genomic level, we performed mRNA expression analyses using Affymetrix microarray gene chips and real-time RT-PCR in sensorimotor cortex of parkinsonian and control rats following STN-HFS. Experimental parkinsonism was induced in Brown Norway rats by bilateral nigral injections of 6-hydroxydopamine and was assessed histologically, behaviorally, and electrophysiologically. We applied prolonged (23h) unilateral STN-HFS in awake and freely moving animals, with the non-stimulated hemisphere serving as an internal control for gene expression analyses. Gene enrichment analysis revealed strongest regulation in major histocompatibility complex (MHC) related genes. STN-HFS led to a cortical downregulation of several MHC class II (RT1-Da, Db1, Ba, and Cd74) and MHC class I (RT1CE) encoding genes. The same set of genes showed increased expression levels in a comparison addressing the effect of 6-hydroxydopamine lesioning. Hence, our data suggest the possible association of altered microglial activity and synaptic transmission by STN-HFS within the sensorimotor cortex of 6-hydroxydopamine treated rats.     

NMR study of non-structural proteins–part III: <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and side-chain resonance assignment of macro domain from <Emphasis Type="Italic">Chikungunya</Emphasis> virus (CHIKV)

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130–190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.     

The genetic basis of triple A (Allgrove) syndrome in a Greek family

Triple A (or Allgrove) syndrome is an autosomal recessive genetic disorder. Patients typically suffer from chronic adrenal insufficiency due to resistance to ACTH (Addison's disease), achalasia of the cardia, and defective tear formation (alacrima). The syndrome is caused by mutations in the AAAS gene which encodes the protein ALADIN, a constituent of eukaryotic nuclear pore complexes. The multi-systemic nature and variable manifestations of the triple A syndrome often confound its diagnosis and limit our understanding of its exact pathogenesis. We performed mutational screening of the AAAS gene in a Greek family of four individuals, including an affected propositus with typical symptoms of late-onset triple A syndrome. Our results are consistent with an autosomal recessive pattern of inheritance within the family, caused by a functional c.43C > A mutation in exon 1 of the AAAS gene. All members of the family were also homozygous for a silent c.855C > T nucleotide change within exon 9 of the AAAS gene, representing a common single nucleotide polymorphism. The compromising c.43C > A mutation is predicted to cause a p.Gln15Lys amino acid substitution in the ALADIN protein. However, it has been suggested that the functional impact of this mutation may be more severe, causing a shift in the reading frame of AAAS gene via formation of an aberrant premature donor splice site within exon 1. We propose that mutational analysis of the AAAS gene should be considered in adult patients with one or more clinical signs of the disease, as diagnosis of late-onset cases can be ambiguous.     

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.