Cardiorespiratory pattern of rest-associated apnea in a Weddell seal: a case study at an ice hole in Antarctica

Seals are known to be periodic breathers with eupneic and apneic phases at rest. We video-recorded a resting Weddell seal (Leptonychotes weddellii) that appeared head up from an ice hole in McMurdo Sound, Antarctica. From the video recorded, the duration of each eupneic and apneic phase was extracted, and heart rate in apneic phase was obtained by counting ripples of the sea surface around the neck of a seal. The results indicated that the distribution of instantaneous heart rate was bimodal. In addition, the higher the apneic heart rate, the shorter the apneic duration as well as the combination of apneic duration and successive eupneic duration (A–E duration). From these analyses, we characterized the periodic breathing of a resting Weddell seal.     

Demonstration of osmotically dependent promotion of aerenchyma formation at different levels in the primary roots of rice using a 'sandwich' method and X-ray computed tomography

Background and Aims

The effect of environmental factors on the regulation of aerenchyma formation in rice roots has been discussed for a long time, because aerenchyma is constitutively formed under aerated conditions. To elucidate this problem, a unique method has been developed that enables sensitive detection of differences in the development of aerenchyma under two different environmental conditions. The method is tested to determine whether aerenchyma development in rice roots is affected by osmotic stress.

Methods

To examine aerenchyma formation both with and without mannitol treatment in the same root, germinating rice (Oryza sativa) caryopses were sandwiched between two agar slabs, one of which contained 270 mm of mannitol. The roots were grown touching both slabs and were thereby exposed unilaterally to osmotic stress. As a non-invasive approach, refraction contrast X-ray computed tomography (CT) using a third-generation synchrotron facility, SPring-8 (Super photon ring 8 GeV, Japan Synchrotron Radiation Research Institute), was used to visualize the three-dimensional (3-D) intact structure of aerenchyma and its formation in situ in rice roots. The effects of unilateral mannitol treatment on the development of aerenchyma were quantitatively examined using conventional light microscopy.

Key Results

Structural continuity of aerenchyma was clearly visualized in 3-D in the primary root of rice and in situ using X-ray CT. Light microscopy and X-ray CT showed that the development of aerenchyma was promoted on the mannitol-treated side of the root. Detailed light microscopic analysis of cross-sections cut along the root axis from the tip to the basal region demonstrated that aerenchyma developed significantly closer to the root tip on the mannitol-treated side of the root.

Conclusions

Continuity of the aerenchyma along the rice root axis was morphologically demonstrated using X-ray CT. By using this ‘sandwich’ method it was shown that mannitol promoted aerenchyma formation in the primary roots of rice.     

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.     

Arabidopsis HDA6 is required for freezing tolerance

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.     

CERT and intracellular trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT has been identified as a key factor for the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains including (i) a START domain capable of catalyzing inter-membrane transfer of ceramide, (ii) a pleckstrin homology domain, which serves to target the Golgi apparatus by recognizing phosphatidylinositol 4-monophosphate, and (iii) a short peptide motif named FFAT motif which interacts with the ER-resident membrane protein VAP. CERT is preferentially distributed to the Golgi region in cells, and Golgi-targeted CERT appears to retain the activity to interact with VAP. On the basis of these results, it has been proposed that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that a particularly efficient cycle of CERT movement for trafficking of ceramide may proceed at membrane contact sites between the ER and the Golgi apparatus.     

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l ‐cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l ‐arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm‐like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non‐covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470–478, 2014     

Exit of GPI‐Anchored Proteins from the ER Differs in Yeast and Mammalian Cells

Previous studies have shown that yeast glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI‐APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI‐APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER‐to‐Golgi transport of GPI‐APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI‐APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI‐APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI‐APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.     

Protein phosphatase 2Cepsilon is an endoplasmic reticulum integral membrane protein that dephosphorylates the ceramide transport protein CERT to enhance its association with organelle membranes

Protein phosphatase 2Cepsilon (PP2Cepsilon), a mammalian PP2C family member, is expressed in various tissues and is implicated in the negative regulation of stress-activated protein kinase pathways. We show that PP2Cepsilon is an endoplasmic reticulum (ER) transmembrane protein with a transmembrane domain at the amino terminus and the catalytic domain facing the cytoplasm. Yeast two-hybrid screening of a human brain library using PP2Cepsilon as bait resulted in the isolation of a cDNA that encoded vesicle-associated membrane protein-associated protein A (VAPA). VAPA is an ER resident integral membrane protein involved in recruiting lipid-binding proteins such as the ceramide transport protein CERT to the ER membrane. Expression of PP2Cepsilon resulted in dephosphorylation of CERT in a VAPA expression-dependent manner, which was accompanied by redistribution of CERT from the cytoplasm to the Golgi apparatus. The expression of PP2Cepsilon also enhanced the association between CERT and VAPA. In addition, knockdown of PP2Cepsilon expression by short interference RNA attenuated the interaction between CERT and VAPA and the sphingomyelin synthesis. These results suggest that CERT is a physiological substrate of PP2Cepsilon and that dephosphorylation of CERT by PP2Cepsilon may play an important role in the regulation of ceramide trafficking from the ER to the Golgi apparatus.