Chemical Studies on “Clover Sickness”

Two new isoflavonoids were isolated from red clover as germination inhibitors for the same plant and their structures were determined as a glucoside of biochanin A (7-d-β-glucosyl-5,7-dihydroxy-4′-methoxyisoflavone) (II) and its 5-malonate (I), respectively. Besides these compounds the following substances were also isolated as inhibitors: trifolirhizin (III), ononin (IV), daidzein (V) and its 7-glucoside (VI), formononetin (VII), genistein (VIII) and biochanin A (IX).     

Environmental perturbations influence telomere dynamics in long-lived birds in their natural habitat

Telomeres are regarded as markers of biological or cellular ageing because they shorten with the degree of stress exposure. Accordingly, telomere lengths should show different rates of change when animals are faced with different intensities of environmental challenges. However, a relationship between telomere length and the environment has not yet been tested within a natural setting. Here, we report longitudinal telomere dynamics in free-living, black-tailed gulls (Larus crassirostris) through the recapture of birds of a known age over 2–5 consecutive years. The rate of change in telomere lengths differed with respect to year but not sex or age. The years when gulls showed stable telomere lengths or increases in telomere lengths (from 2009 to 2010) and decreases in telomere lengths (from 2010 to 2011) were characterized by El Niño and the Great Japan Earthquake, respectively. Both events are suspected to have had long-lasting effects on food availability and/or weather conditions. Thus, our findings that telomere dynamics in long-lived birds are influenced by dramatic changes in environmental conditions highlight the importance of environmental fluctuations in affecting stress and lifespan.     

Prolonged post-hyperventilation apnea in two young adults with hyperventilation syndrome

Background

The prognosis of hyperventilation syndrome (HVS) is generally good. However, it is important to proceed with care when treating HVS because cases of death following hyperventilation have been reported. This paper was done to demonstrate the clinical risk of post-hyperventilation apnea (PHA) in patients with HVS.

Case presentation

We treated two patients with HVS who suffered from PHA. The first, a 21-year-old woman, had a maximum duration of PHA of about 3.5?minutes and an oxygen saturation (SpO₂) level of 60%. The second patient, a 22-year-old woman, had a maximum duration of PHA of about 3?minutes and an SpO₂ level of 66%. Both patients had loss of consciousness and cyanosis. Because there is no widely accepted regimen for treating patients with prolonged PHA related to HVS, we administered artificial ventilation to both patients using a bag mask and both recovered without any after effects.

Conclusion

These cases show that some patients with HVS develop prolonged PHA or severe hypoxia, which has been shown to lead to death in some cases. Proper treatment must be given to patients with HVS who develop PHA to protect against this possibility. If prolonged PHA or severe hypoxemia arises, respiratory assistance using a bag mask must be done immediately.

     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2–PTEN–AKT Pathway and Placental Cell Proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2–PTEN–AKT pathway and placental cell proliferation.     

Loss of the N-acetylgalactosamine side chain of the GPI-anchor impairs bone formation and brain functions and accelerates the prion disease pathology

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrP^C, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrP^C GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.     

Relationship Between Amino Acid Properties and Protein Stability: Buried Mutations

In order to understand the mechanism of protein stability and to develop a simple method for predicting mutation-induced stability changes, we analyzed the relationship between stability changes caused by buried mutations and changes in 48 amino acid properties. As expected from the importance of hydrophobicity, properties reflecting hydrophobicity are strongly correlated with the stability of proteins. We found that subgroup classification based on secondary structure increased correlations significantly, and mutations within -strand segments correlated better than did those in -helical segments, which may result from stronger hydrophobicity of the -strands. Multiple regression analyses incorporating combinations of three properties from among all possible combinations of the 48 properties increased the correlation coefficient to 0.88 and by an average of 13% for all data sets. Analyzing the stability of tryptophan synthase mutants with Glu49 replaced by all other residues except Arg revealed that combining buriedness, solvent-accessible surface area for denatured protein, and unfolding Gibbs free energy change increased the correlation to 0.95. Consideration of sequence and structural information (neighboring residues in sequence and in space) did not significantly strengthen the correlations in buried mutations, suggesting that nonspecific interactions dominate in the interior of proteins.