Mechanical Properties of the Icosahedral Shell of Southern Bean Mosaic Virus: A Molecular Dynamics Study

The mechanical properties of viral shells are crucial for viral assembly and infection. To study their distribution and heterogeneity on the viral surface, we performed atomistic force-probe molecular dynamics simulations of the complete shell of southern bean mosaic virus, a prototypical T = 3 virus, in explicit solvent. The simulation system comprised more than 4,500,000 atoms. To facilitate direct comparison with atomic-force microscopy (AFM) measurements, a Lennard-Jones sphere was used as a model of the AFM tip, and was pushed with different velocities toward the capsid protein at 19 different positions on the viral surface. A detailed picture of the spatial distribution of elastic constants and yielding forces was obtained that can explain corresponding heterogeneities observed in previous AFM experiments. Our simulations reveal three different deformation regimes: a prelinear regime of outer surface atom rearrangements, a linear regime of elastic capsid deformation, and a rearrangement regime that describes irreversible structural changes and the transition from elastic to plastic deformation. For both yielding forces and elastic constants, a logarithmic velocity dependency is evident over nearly two decades, the explanation for which requires including nonequilibrium effects within the established theory of enforced barrier crossing.     

Reduced Expression of GABA Transporter GAT3 in Helpless Rats,an Animal Model of Depression

Mood disorders have been linked to glial and synaptic pathology such as disturbed neurotransmission of γ-aminobutyric acid (GABA). We evaluated the expression of GABAergic marker genes in rats with helpless behaviour, an animal model of depression. Male Sprague-Dawley rats from inbred lines were tested for helpless behaviour and grouped according to failures in terminating foot shock currents. Expression levels of GABAergic marker genes were assessed using semiquantitative in situ-hybridization. Animals with congenital helpless behaviour (cH) were unable to escape current exposure in contrast to cH-animals derived from the same litters with low failure rates and to non-helpless animals (cNH). We found a significant downregulation of the GABA transporter GAT3 in cLH rats. GAT1 showed small changes, glutamic acid decarboxylase (GAD67) and the vesicular GABA transporter were not significantly altered. Reduced GABA transporter expression is well in concert with the behavioural phenotypes of knockout animals and strengthens the hypothesis of impaired glial functions in depression.     

Lesion bypass of N2-ethylguanine by human DNA polymerase iota

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.     

Loss of Parkin or PINK1 Function Increases Drp1-dependent Mitochondrial Fragmentation

Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.Many lines of evidence suggest that mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson disease, starting from the early observation that the complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced acute and irreversible parkinsonism in young drug addicts (for review, see Refs. 1–3). In support of a crucial role of mitochondria in Parkinson disease, several Parkinson disease-associated gene products directly or indirectly impinge on mitochondrial integrity (for review, see Refs. 4–6). A clear link between Parkinson disease genes and mitochondria has recently emerged from studies on PINK1 (PTEN-induced putative kinase 1), a mitochondrial serine/threonine kinase, and parkin, a cytosolic E3 ubiquitin ligase. Drosophila parkin null mutants displayed reduced life span, male sterility, and locomotor defects due to apoptotic flight muscle degeneration (7). The earliest manifestation of muscle degeneration and defective spermatogenesis was mitochondrial pathology, exemplified by swollen mitochondria and disintegrated cristae. Remarkably, Drosophila PINK1 null mutants shared marked phenotypic similarities with parkin mutants, and parkin could compensate for the PINK1 loss-of-function phenotype but not vice versa, leading to the conclusion that PINK1 and parkin function in a common genetic pathway with parkin acting downstream of PINK1 (8–10). We recently demonstrated that PINK1 deficiency in cultured human cells causes alterations in mitochondrial morphology, which can be rescued by wild type parkin but not by pathogenic parkin mutants (11). We now present evidence that parkin plays an essential role in maintaining mitochondrial integrity. RNAi³-mediated knockdown of parkin increases mitochondrial fragmentation and decreases cellular ATP production. Notably, mitochondrial fragmentation induced by PINK1/parkin deficiency is observed not only in human neuroblastoma cells but also in primary mouse neurons and insect S2 cells. Alterations in mitochondrial morphology are early manifestations of parkin/PINK1 silencing that are not caused by an increase in apoptosis. The mitochondrial phenotype observed in parkin- or PINK1-deficient cells can morphologically and functionally be rescued by the increased expression of a dominant negative mutant of the fission-promoting protein Drp1. Moreover, manifestation of the PINK1/parkin knockdown phenotype is dependent on Drp1 expression, indicating that an acute loss of parkin or PINK1 function increases mitochondrial fission.     

Tec‐Kinase‐Mediated Phosphorylation of Fibroblast Growth Factor 2 is Essential for Unconventional Secretion

Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi‐independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5‐biphosphate (PI(4,5)P₂) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post‐translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.     

The co-chaperone SGT of <Emphasis Type="Italic">Leishmania donovani</Emphasis> is essential for the parasite's viability

Molecular chaperone proteins play a pivotal role in the protozoan parasite Leishmania donovani, controlling cell fate and ensuring intracellular survival. In higher eukaryotes, the so-called co-chaperone proteins are required for client protein recognition and proper function of chaperones, among them the small glutamine-rich tetratricopeptide repeat proteins (SGT) which interact with both HSP70 and HSP90 chaperones. An atypical SGT homolog is found in the L. donovani genome, encoding a protein lacking the C-terminal glutamine-rich region, normally typical for SGT family members. The gene is expressed constitutively during the life cycle and is essential for survival and/or growth of the parasites. LdSGT forms large, stable complexes that also include another putative co-chaperone, HSC70 interacting protein (HIP). The gene product forms cytoplasmic clusters, matching the subcellular distribution of HIP and partly that of the major cytoplasmic chaperones, HSP70 and HSP90, reflecting a direct molecular interaction with both chaperones.     

Barn swallows before barns: population histories and intercontinental colonization

The barn swallow (Hirundo rustica) is one of most widely distributed swallows, owing in part to its recent switch from natural nest sites to human structures. We conducted phylogenetic analysis of mitochondrial (mt) and nuclear DNA to explore the recent evolutionary history of this species. Strongly supported mtDNA clades corresponded to Europe, Asia and North America plus the Baikal region of Asia. Analysis of sequence data from a sex-linked nuclear gene was unable to recover the phylogenetic splits in the mtDNA tree, confirming that the main clades evolved recently. The phylogenetic pattern suggests that the ancestral area of the barn swallow was the holarctic; most divergence events are consistent with vicariance. Most unexpectedly, analyses show that barn swallows from North America colonized the Baikal region in the recent past (one fixed substitution). This dispersal direction is opposite of that for most nearctic-palearctic taxon exchanges. Although this invasion was envisioned to coincide with the appearance of new types of human dwelling in the Baikal region, calibration of molecular divergence suggests an older dispersal event. A recent history of gene flow within the main palearctic clades is consistent with range and population expansion owing to new nesting opportunities provided by human settlements. Contrary to expectation, populations in North America appear historically larger and more stable than those in the palearctic. The Baikal population apparently has not increased greatly since colonization.     

Parturition in horses is dominated by parasympathetic activity of the autonomous nervous system

External and internal stressors prolong parturition in different species. At parturition, sympathoadrenal activation should be avoided because an increased sympathetic tone may cause uterine atonia via β₂-receptors. We hypothesized that at physiological parturition, horses are under parasympathetic dominance, and stress-response mechanisms are not activated during delivery of the foal. To evaluate stress responses, heart rate, heart rate variability, catecholamines, and cortisol were analyzed in mares (n = 17) throughout foaling. Heart rate decreased from 2 hours before (51 ± 1 beats/minute) to 2 hours after delivery (41 ± 2 beats/minute; P < 0.05). Heart rate variability variables, standard deviation of the beat-to-beat interval, and root mean square of successive beat-to-beat differences, changed over time (P < 0.05) with the highest values within 15 minutes after delivery. The number of mares with atrioventricular blocks and the number of atrioventricular blocks per mare increased over time (P < 0.01) and were significantly elevated from 15 minutes before to 45 minutes after birth of the foal. Salivary cortisol concentrations increased to a maximum at 30 minutes after delivery (25.0 ± 3.4 ng/mL; P < 0.01). Plasma epinephrine and norepinephrine concentrations showed significant fluctuations from rupture of the allantochorion to expulsion of the fetal membranes (P < 0.01) but were not markedly elevated at any time. In conclusion, mares give birth under high parasympathetic tone. Cortisol release during and after foaling is most likely part of the endocrine pathways regulating parturition and not a labor-associated stress response.