Sustained torpidity following multi-dose administration of 3-iodothyronamine in mice

Despite significant medical benefits as in space exploration or emergency care, prolonged torpidity of non-hibernator mammals remains unexplored to date. Here, we report that male Institute of Cancer Research mice could sustain two separate 2-day torpor bouts and maintain body temperature of 28-33°C following repeated treatments of 3-iodothyronamine (T(1) AM), a natural derivative of thyroid hormone. A 1-day interbout arousal period, adopted to mimic the behavior of true hibernators, seemed critical for the subjects to restore physiological homeostasis. Molecular studies of neuron-specific enolase, S100 calcium binding protein B and heat shock protein 72 suggested that the brain maintains functional and cytoprotective activities during sustained torpidity. Together, the results of this study propose a practical protocol using a torpor-arousal cycle that can be applied to the extreme medical situations.     

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.     

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).     

Salicylic acid treatment of pea seeds induces its de novo synthesis

Salicylic acid (SA), which is known as a signal molecule in the induction of defense mechanisms in plants, could be a promising compound for the reduction of stress sensitivity. The aim of the present work was to investigate the distribution of SA in young pea (Pisum sativum L.) seedlings grown from seeds soaked in ³H-labeled SA solution before sowing, and to study the physiological changes induced by this seed treatment. The most pronounced changes in SA levels occurred in the epicotyl and the seeds. Radioactivity was detected only in the bound form of SA, the majority of which was localized in the seeds, and only a very low level of radioactivity was detected in the epicotyl. SA pre-treatment increased the expression of the chorismate synthase and isochorismate synthase genes in the epicotyl. Pre-soaking the seeds in SA increased the activities of some antioxidant enzymes, namely ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) and the level of ortho-hydroxycinnamic acid, but decreased the level of polyamines. These results suggest that the increased level of free and bound SA detected in plants growing from seeds soaked in SA solution before sowing is the product of de novo synthesis, rather than having been taken up and mobilized by the plants.     

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT_So, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT_So represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.     

Contrasting dynamics of radial O2-loss barrier induction and aerenchyma formation in rice roots of two lengths

Background and Aims

Many wetland species form aerenchyma and a barrier to radial O₂ loss (ROL) in roots. These features enhance internal O₂ diffusion to the root apex. Barrier formation in rice is induced by growth in stagnant solution, but knowledge of the dynamics of barrier induction and early anatomical changes was lacking.

Methods

ROL barrier induction in short and long roots of rice (Oryza sativa L. ‘Nipponbare’) was assessed using cylindrical root-sleeving O₂ electrodes and methylene blue indicator dye for O₂ leakage. Aerenchyma formation was also monitored in root cross-sections. Microstructure of hypodermal/exodermal layers was observed by transmission electron microscopy (TEM).

Key Results

In stagnant medium, barrier to ROL formation commenced in long adventitious roots within a few hours and the barrier was well formed within 24 h. By contrast, barrier formation took longer than 48 h in short roots. The timing of enhancement of aerenchyma formation was the same in short and long roots. Comparison of ROL data and subsequent methylene blue staining determined the apparent ROL threshold for the dye method, and the dye method confirmed that barrier induction was faster for long roots than for short roots. Barrier formation might be related to deposition of new electron-dense materials in the cell walls at the peripheral side of the exodermis. Histochemical staining indicated suberin depositions were enhanced prior to increases in lignin.

Conclusions

As root length affected formation of the barrier to ROL, but not aerenchyma, these two acclimations are differentially regulated in roots of rice. Moreover, ROL barrier induction occurred before histochemically detectable changes in putative suberin and lignin deposits could be seen, whereas TEM showed deposition of new electron-dense materials in exodermal cell walls, so structural changes required for barrier functioning appear to be more subtle than previously described.     

Identification and evolution of venom phospholipase A2 inhibitors from Protobothrops elegans serum

The cDNAs encoding venom phospholipase A(2) (PLA(2)) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A(a) and PeαPLI-A(b), were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA(2)s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA(2) isozymes.     

Calcium binding protein expression in the optic tectum of Alligator during development

The onset and distribution of the calcium binding proteins, calretinin, calbindin, and parvalbumin, were examined in the optic tectum of Alligator mississipiensis embryos between Stages 18 and 26–28. The immunoreactivity of each calcium binding protein correlated well with the results from the Western blot experiments. In terms of onset and distribution, calretinin expressison was the most widespread of the three calcium binding proteins that were examined, and was also the earliest to be visualized. Calbindin expression occurred next, whereas parvalbumin expression was the most limited and appeared last. For small calretinin (+) neurons, the pattern of immunoreactivity during development was from inside to outside, whereas for the larger cells, it was from outside to inside. For calbindin immunoreactive cells in the superficial zone, the pattern was from outside to inside. The distribution of the parvalbumin immunopositive neurons did not change significantly over the time period examined. Similar data on other amniotes is limited. However, the pattern in Alligator shares some similarities with kittens in regards to the distribution of calbindin and parvalbumin in the developing superior colliculus. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 899–910, 2013