Subcellular Distribution of Calpain and Calpastatin Immunoreactivity and Fodrin Proteolysis in Rabbit Hippocampus After Hypoxia and Glucocorticoid Treatment

Abstract: Rabbits were subjected to hypoxia (5% O₂) for up to 90 min and allowed to recover for a maximum of 4 days. Hippocampus homogenate was assayed for fodrin breakdown product (BDP). After separation into a nuclear and mitochondrial fraction (NMF), a membrane and microsomal fraction (MMF), and a cytosolic fraction (CF), samples were assayed for μ-calpain, m-calpain, and calpastatin immunoreactivity. Calpain and calpastatin immunoreactivity decreased in the NMF and CF but increased in the MMF during hypoxia and short-term recovery. This translocation occurred in parallel with the increase in fodrin BDP. Because the increase in the MMF was not large enough to explain the decrease in the other two fractions, it was assumed that the translocation and activation was accompanied by a reduction in the total amounts of calpains and calpastatin. Glucocorticoid pretreatment (beta-methasone, 0.4 mg × kg⁻¹× day⁻¹) for 7 days produced a decrease in the ratio of activated μ-calpain in all three fractions in nearly all samples before, during, and after hypoxia, compared with untreated animals. Glucocorticoid pretreatment also prevented the increase in fodrin BDP that occurred in untreated animals during hypoxia and short-term recovery, indicating impairment of calpain activation.     

Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

Summary A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYIN-LITRQRY-NH₂, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15°C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined -helix in the centre, with a few residues at the termini having less well defined conformations. The spinlattice and spin-spin relaxation rates of -carbons have been determined on ¹³C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C-H vectors in the -helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.Supplementary material available from the authors: Two data tables and 10 PDB coordinate files of the calculated NMR structures of P7. One data table contains all detected and integrated NOE intensities; the other connects each proton and pseudoatom to an atom number used in the NOE table. The table contents served as input data files for CALIBA.Currently on leave from the Institute of Chemical Physics and Biophysics, Tallinn, Estonia.     

Do Fleas,and/or Old Nest Material,Influence Nest-site preference in Hole-nesting Passerines?

This study examined nest-site choice in a migratory population of pied flycatchers (Ficedula hypoleuca) and sympatric populations of three resident tit species (Parus major, P. caeruleus and P. palustris) in central Sweden. All four species are secondary-cavity nesters which naturally breed in pre-formed tree cavities but readily use artificial nest boxes. We asked whether flycatchers and tits discriminate between nest boxes that: 1. Are ‘empty’; 2. Contain old nests without ectoparasites (fleas Ceratophyllus sp.); or 3. Contain old nests with ectoparasites. We found that pied flycatchers preferred nest boxes containing old nests, regardless of whether these nests held parasites. In contrast, tits did not discriminate between the three types of boxes. Tits may pay a cost for their lack of choosiness: after the breeding season, tit nests contained more fleas than flycatcher nests. Nevertheless, parasites did not affect the choice of a nest site in any of the species studied. We suggest that the migratory flycatchers are in a hurry to start breeding upon arrival and use the presence of an old nest as a shortcut cue to assess nest-site quality. Also, they may save valuable time by copying the choice of previous breeders. Non-migratory tits may have more time to inspect nest sites, but do not seem to use the same cues in nest-site selection as the pied flycatcher.     

Molecular Phylogeny and Evolution of the Neurotrophins from Monotremes and Marsupials

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree. Received: 27 January 1997 / Accepted: 20 March 1997     

Does Social Isolation and Low Societal Participation Predict Disability Pension? A Population Based Study

Purpose

The aim was to examine the potential influence of social isolation and low societal participation on the future risk of receiving disability pension among individuals in Sweden. A specific aim was to describe differences depending on disability pension diagnoses, and how the results were modified by sex and age.

Method

The study comprised representative samples of Swedish women and men, who had been interviewed in any of the annual Swedish Surveys of Living Conditions between 1990 and 2007. Information on disability pension and diagnoses was added from the Swedish Social Insurance Agency’s database (1991-2011). The mean number of years of follow-up for the 53920 women and men was twelve years (SD 5.5), and the study base was restricted to the ages 20 to 64 years of age. The predictors were related to disability pension by Cox’s proportional hazards regression.

Results

Social isolation and low societal participation were associated with future disability pension also after control for age, year of interview, socio demographic conditions and self reported longstanding illness. Lone individuals were at increased risk of disability pension, and the effect of living without children was modified by sex and age. An increase in risk was particularly noticeable among younger women who reported that they had sparse contacts with others, and no close friend. Both women and men who reported that they did not participate in political discussions and who could not appeal on a decision by a public authority were also at increased risk. The effects of social isolation were mainly attributed to disability pension with mental diagnoses, and to younger individuals.

Conclusions

The study suggests that social isolation and low societal participation are predictors of future disability pension. Social isolation and low societal participation increased particularly the risk of future disability pension in mental diagnoses among younger individuals.     

Frequency Dependence of Signal Power and Spatial Reach of the Local Field Potential

Despite its century-old use, the interpretation of local field potentials (LFPs), the low-frequency part of electrical signals recorded in the brain, is still debated. In cortex the LFP appears to mainly stem from transmembrane neuronal currents following synaptic input, and obvious questions regarding the ‘locality’ of the LFP are: What is the size of the signal-generating region, i.e., the spatial reach, around a recording contact? How far does the LFP signal extend outside a synaptically activated neuronal population? And how do the answers depend on the temporal frequency of the LFP signal? Experimental inquiries have given conflicting results, and we here pursue a modeling approach based on a well-established biophysical forward-modeling scheme incorporating detailed reconstructed neuronal morphologies in precise calculations of population LFPs including thousands of neurons. The two key factors determining the frequency dependence of LFP are the spatial decay of the single-neuron LFP contribution and the conversion of synaptic input correlations into correlations between single-neuron LFP contributions. Both factors are seen to give low-pass filtering of the LFP signal power. For uncorrelated input only the first factor is relevant, and here a modest reduction (<50%) in the spatial reach is observed for higher frequencies (>100 Hz) compared to the near-DC () value of about . Much larger frequency-dependent effects are seen when populations of pyramidal neurons receive correlated and spatially asymmetric inputs: the low-frequency () LFP power can here be an order of magnitude or more larger than at 60 Hz. Moreover, the low-frequency LFP components have larger spatial reach and extend further outside the active population than high-frequency components. Further, the spatial LFP profiles for such populations typically span the full vertical extent of the dendrites of neurons in the population. Our numerical findings are backed up by an intuitive simplified model for the generation of population LFP.     

Hagfish phylogeny and taxonomy,with description of the new genus Rubicundus (Craniata,Myxinidae)

A recent phylogenetic analysis of the Myxinidae based on the 16S rRNA gene resulted in synonymization of Paramyxine with Eptatretus. This created homonymy of Paramyxine fernholmi with Eptatretus fernholmi and Paramyxine wisneri with Eptatretus wisneri. In order to resolve this nomenclatural dilemma, we made a more extensive phylogenetic assessment of the Myxinidae and examined the nomenclature of the family. We used 75 sequences (37 of which new for this study) of a 561 bp fragment of the 16S rRNA gene, representing 33 species, and 72 sequences (37 of which new for this study) of a 687 bp fragment of the cytochrome c oxidase subunit I (COI) gene, representing 23 species, to reconstruct the phylogeny of Myxinidae. The monophyly of the subfamily Myxininae, traditionally characterized by having a single pair of external gill openings, was rejected (0.50 Bayesian posterior probability) by the 16S analysis, but supported by the COI and combined COI+16S analyses (0.99 and 0.81 Bpp, respectively). The monophyly of the subfamily Eptatretinae, characterized by having several pairs of external gill openings, was not supported by the 16S analysis and rejected by the COI and combined COI+16S analysis due to the placement of Eptatretus lopheliae as the earliest branch of Myxinidae (0.71 and 0.57 Bpp, respectively). Eptatretus lopheliae and Eptatretus rubicundus formed a monophyletic group and were allocated to a new genus, Rubicundus, characterized by the presence of an elongated tubular nostril and reddish coloration. A new monotypic subfamily, Rubicundinae, was proposed for Rubicundus. The synonymy of the genera Paramyxine and Quadratus with Eptatretus was confirmed. E. fernholmi is renamed Eptatretus luzonicus. Eptatretus wisneri was renamed Eptatretus bobwisneri. Petromyzon cirrhatus Forster, 1801, Homea banksii Fleming, 1822, and Bdellostoma forsteri Müller, 1836 are synonyms, but no type specimens are known to exist. Petromyzon cirrhatus was designated as type species of Eptatretus, conserving present usage. Gastrobranchus dombeyi Shaw, 1804 has priority over other names for Chilean myxinids. Bdellostoma stoutii was designated as type species of Polistotrema Gill. The validity of the Western Atlantic Myxine limosa as distinct from the Eastern Atlantic Myxine glutinosa was confirmed.     

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.     

Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2)

Streptomycetes grow by cell wall extension at hyphal tips. The molecular basis for such polar growth in prokaryotes is largely unknown. It is reported here that DivIVASC, the Streptomyces coelicolor homologue of the Bacillus subtilis protein DivIVA, is essential and directly involved in hyphal tip growth and morphogenesis. A DivIVASC-EGFP hybrid was distinctively localized to hyphal tips and lateral branches. Reduction of divIVASC expression to about 10% of the normal level produced a phenotype strikingly similar to that of many tip growth mutants in fungi, including irregular curly hyphae and apical branching. Overexpression of the gene dramatically perturbed determination of cell shape at the growing tips. Furthermore, staining of nascent peptidoglycan with a fluorescent vancomycin conjugate revealed that induction of overexpression in normal hyphae disturbed tip growth, and gave rise to several new sites of cell wall assembly, effectively causing hyperbranching. The results show that DivIVASC is a novel bacterial morphogene, and it is localized at or very close to the apical sites of peptidoglycan assembly in Streptomyces hyphae.