Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage.

Lipin 1 is an ER enzyme that produces diacylglycerol, the lipid intermediate that feeds into the synthesis of glycerophospholipids for membrane expansion or triacylglycerol for storage into lipid droplets. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but a role for CTDNEP1 in lipid storage in mammalian cells is not known. Here, we show that CTDNEP1 is reliant on its binding partner NEP1R1 for its stability and function in limiting ER expansion. CTDNEP1 contains an amphipathic helix at its N-terminus that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 and NEP1R1 and show that they facilitate complex formation in vivo and in vitro. We demonstrate that NEP1R1 binding to CTDNEP1 shields CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, we found that NEP1R1 is not required for CTDNEP1's role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on NEP1R1 depends on cellular demands for membrane production versus lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis under different metabolic conditions.

Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.

Comparative differences in maintaining membrane fluidity and remodeling cell wall between Glycine soja and Glycine max leaves under drought.

Water shortage is one of the most important environmental factors limiting crop yield. In this study, we used wild soybean (Glycine soja Sieb. et Zucc.) and soybean (Glycinemax (L.) Merr.) seedlings as experimental materials, simulated drought stress using soil gravimetry, measured growth and physiological parameters, and analyzed differentially expressed genes and metabolites in the leaves of seedling by integrated transcriptomics and metabolomics techniques. The results indicate that under water deficit, Glycine soja maintained stable photosynthate by accumulating Mg2+, Fe3+, Mn2+, Zn2+ and B3+, and improved water absorption by increasing root growth. Notably, Glycine soja enhanced linoleic acid metabolism and plasma membrane intrinsic protein (PIP1) gene expression to maintain membrane fluidity, and increased pentose, glucuronate and galactose metabolism and thaumatin protein genes expression to remodel the cell wall, thereby increasing water-absorption to better tolerate to drought stress. In addition, it was found that secondary phenolic metabolism, such as phenylpropane biosynthesis, flavonoid biosynthesis and ascobate and aldarate metabolism were weakened, resulting in the collapse of the antioxidant system, which was the main reason for the sensitivity of Glycine max to drought stress. These results provide new insights into plant adaptation to water deficit and offer a theoretical basis for breeding soybean varieties with drought tolerance.

Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a non-canonical protein serine/threonine phosphatase that regulates ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of CTDNEP1 or NEP1R1 in human cells generate identical phenotypes, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a pseudo-substrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.

Effects of Tremella fuciformis Mushroom Polysaccharides on Structure, Pasting, and Thermal Properties of Chinese Chestnuts (Castanea henryi) Starch Granules under Different Freeze-Thaw Cycles.

The purpose of this study was to investigate the effect of Tremella fuciformis polysaccharides on the physicochemical properties of freeze-thawed cone chestnut starch. Various aspects, including water content, crystallinity, particle size, gelatinization, retrogradation, thermal properties, rheological properties, and texture, were examined. The results revealed that moderate freezing and thawing processes increased the retrogradation of starch; particle size, viscosity, shear type, hinning degree, and hardness decreased. After adding Tremella fuciformis polysaccharide, the particle size, relative crystallinity, and gelatinization temperature decreased, which showed solid characteristics. Consequently, the inclusion of Tremella fuciformis polysaccharide effectively countered dehydration caused by freezing and thawing, reduced viscosity, and prevented the retrogradation of frozen-thawed chestnut starch. Moreover, Tremella fuciformis polysaccharide played a significant role in enhancing the stability of the frozen-thawed chestnut starch. These findings highlight the potential benefits of incorporating Tremella fuciformis polysaccharides in starch-based products subjected to freeze-thaw cycles.

Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage.

The endoplasmic reticulum (ER) is the site for the synthesis of the major membrane and storage lipids. Lipin 1 produces diacylglycerol, the lipid intermediate critical for the synthesis of both membrane and storage lipids in the ER. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but its role in lipid storage in mammalian cells is unknown. Here, we show that the ubiquitin-proteasome degradation pathway controls the levels of ER/nuclear envelope-associated CTDNEP1 to regulate ER membrane synthesis through lipin 1. The N-terminus of CTDNEP1 is an amphipathic helix that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 with its regulatory subunit NEP1R1 and show that they facilitate complex formation in vivo and in vitro . We demonstrate a role for NEP1R1 in temporarily shielding CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, we found that NEP1R1 is not required for CTDNEP1's role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on its regulatory subunit differs during ER membrane synthesis and lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis and storage under fluctuating conditions.

Comparative study of leaf nutrient reabsorption by two different ecotypes of wild soybean under low-nitrogen stress.

Wild soybean (Glycine soja), the ancestor of cultivated soybean, has evolved into many ecotypes with different adaptations to adversity under the action of divergent evolution. Barren-tolerant wild soybean has developed adaptation to most nutrient-stress environments, especially with respect to low nitrogen (LN) conditions. This study describes the differences in physiological and metabolomic changes between common wild soybean (GS1) and barren-tolerant wild soybean(GS2) under LN stress. Compared with plants grown under the unstressed control (CK) conditions, the young leaves of barren-tolerant wild soybean under LN conditions maintained relatively stable chlorophyll, concentration and rates of photosynthesis and transpiration, as well as increased carotenoid content, whereas the net photosynthetic rate (PN) of GS1 decreased significantly 0.64-fold (p < 0.05) in the young leaves of GS1. The ratio of internal to atmospheric CO2 concentrations increased significantly 0.07-fold (p < 0.05), 0.09-fold (p < 0.05) in the young leaves of GS1 and GS2, respectively, and increased significantly 0.05-fold (p < 0.05) and 0.07-fold (p < 0.05) in the old leaves of GS1 and GS2, respectively, relative to the CK. The concentration of chlorophylls a and b decreased significantly 0.45-fold (p < 0.05), 0.13-fold (p > 0.05) in the young leaves of GS1 and GS2, respectively, and decreased significantly 0.74-fold (p < 0.01) and 0.60-fold (p < 0.01) in the old leaves of GS1 and GS2, respectively. Under LN stress, nitrate concentration in the young leaves of GS1 and GS2 decreased significantly 0.69- and 0.50-fold (p < 0.01), respectively, relative to CK, and decreased significantly 2.10-fold and 1.77-fold (p < 0.01) in the old leaves of GS1 and GS2, respectively. Barren-tolerant wild soybean increased the concentration of beneficial ion pairs. Under LN stress, Zn2+ significantly increased by 1.06- and 1.35-fold (p < 0.01) in the young and old leaves of GS2 (p < 0.01), but there was no significant change in GS1. The metabolism of amino acids and organic acids was high in GS2 young and old leaves, and the metabolites related to the TCA cycle were significantly increased. The 4-aminobutyric acid (GABA) concertation decreased significantly 0.70-fold (p < 0.05) in the young leaves of GS1 but increased 0.21-fold (p < 0.05) significantly in GS2. The relative concentration of proline increased significantly 1.21-fold (p < 0.01) and 2.85-fold (p < 0.01) in the young and old leaves of GS2. Under LN stress, GS2 could maintain photosynthesis rate and enhance the reabsorption of nitrate and magnesium in young leaves, compared to GS1. More importantly, GS2 exhibited increased amino acid and TCA cycle metabolism in young and old leaves. Adequate reabsorption of mineral and organic nutrients is an important strategy for barren-tolerant wild soybeans to survive under LN stress. Our research provides a new perspective on the exploitation and utilization of wild soybean resources.

Integrated metabolomic and transcriptomic strategies to reveal alkali-resistance mechanisms in wild soybean during post-germination growth stage.

MAIN CONCLUSION: The keys to alkali-stress resistance of barren-tolerant wild soybean lay in enhanced reutilization of reserves in cotyledons as well as improved antioxidant protection and organic acid accumulation in young roots. Soil alkalization of farmlands is increasingly serious, adversely restricting crop growth and endangering food security. Here, based on integrated analysis of transcriptomics and metabolomics, we systematically investigated changes in cotyledon weight and young root growth in response to alkali stress in two ecotypes of wild soybean after germination to reveal alkali-resistance mechanisms in barren-tolerant wild soybean. Compared with barren-tolerant wild soybean, the dry weight of common wild soybean cotyledons under alkali stress decreased slowly and the length of young roots shortened. In barren-tolerant wild soybean, nitrogen-transport amino acids asparagine and glutamate decreased in cotyledons but increased in young roots, and nitrogen-compound transporter genes and genes involved in asparagine metabolism were significantly up-regulated in both cotyledons and young roots. Moreover, isocitric, succinic, and L-malic acids involved in the glyoxylate cycle significantly accumulated and the malate synthetase gene was up-regulated in barren-tolerant wild soybean cotyledons. In barren-tolerant wild soybean young roots, glutamate and glycine related to glutathione metabolism increased significantly and the glutathione reductase gene was up-regulated. Pyruvic acid and citric acid involved in pyruvate-citrate metabolism increased distinctly and genes encoding pyruvate decarboxylase and citrate synthetase were up-regulated. Integrated analysis showed that the keys to alkali-stress resistance of barren-tolerant wild soybean lay in enhanced protein decomposition, amino acid transport, and lipolysis in cotyledons as well as improved antioxidant protection and organic acid accumulation in young roots. This study provides new ideas for the exploitation and utilization of wild soybean resources.

Chemical Bonding and Dynamic Structural Fluxionality of a Boron-Based Na5B7 Sandwich Cluster.

Doping alkali metals into boron clusters can effectively compensate for the intrinsic electron deficiency of boron and lead to interesting boron-based binary clusters, owing to the small electronegativity of the former elements. We report on the computational design of a three-layered sandwich cluster, Na5B7, on the basis of global-minimum (GM) searches and electronic structure calculations. It is shown that the Na5B7 cluster can be described as a charge-transfer complex: [Na4]2+[B7]3-[Na]+. In this sandwich cluster, the [B7]3- core assumes a molecular wheel in shape and features in-plane hexagonal coordination. The magic 6π/6σ double aromaticity underlies the stability of the [B7]3- molecular wheel, following the (4n + 2) Hückel rule. The tetrahedral Na4 ligand in the sandwich has a [Na4]2+ charge-state, which is the simplest example of three-dimensional aromaticity, spherical aromaticity, or superatom. Its 2σ electron counting renders σ aromaticity for the ligand. Overall, the sandwich cluster has three-fold 6π/6σ/2σ aromaticity. Molecular dynamics simulation shows that the sandwich cluster is dynamically fluxional even at room temperature, with a negligible energy barrier for intramolecular twisting between the B7 wheel and the Na4 ligand. The Na5B7 cluster offers a new example for dynamic structural fluxionality in molecular systems.

Chemical bonding and dynamic structural fluxionality of a boron-based Al2B8 binary cluster: the robustness of a doubly 6π/6σ aromatic [B8]2- molecular wheel.

Despite the isovalency between Al and B elements, Al-doping in boron clusters can deviate substantially from an isoelectronic substitution process. We report herein on a unique sandwich di-Al-doped boron cluster, Al2B8, using global structural searches and quantum chemical calculations. The cluster features a perfectly planar B8 molecular wheel, with two isolated Al atoms symmetrically floating above and below it. The two Al atoms are offset from the center of the molecular wheel, resulting in a C 2v symmetry for the cluster. The Al2B8 cluster is shown to be dynamically fluxional even at far below room temperature (100 K), in which a vertical Al2 rod slides or rotates freely within a circular rail on the B8 plate, although there is no direct Al-Al interaction. The energy barrier for intramolecular rotation is only 0.01 kcal mol-1 at the single-point CCSD(T) level. Chemical bonding analysis shows that the cluster is a charge-transfer complex and can be formulated as [Al]+[B8]2-[Al]+. The [B8]2- molecular wheel in sandwich cluster has magic 6π/6σ double aromaticity, which underlies the dynamic fluxionality, despite strong electrostatic interactions between the [Al]+, [B8]2-, and [Al]+ layers.

Transcriptome analysis reveals the mechanisms of hepatic injury caused by long-term environmental exposure to atrazine in juvenile common carp (Cyprinus carpio L.).

Atrazine (ATZ) is the second most commonly used herbicide worldwide, resulting in the pollution of water bodies and affecting the economic benefits of aquaculture. ATZ is known to cause liver damage in the common carp, Cyprinus carpio L., one of the most widely cultivated fish in China, but the underlying mechanisms are poorly understood. In this study, juvenile common carp Cyprinus carpio L. were exposed to three different environmental levels (0.4, 0.8, and 1.2 µg/L) of ATZ for 12 weeks and changes in the liver transcriptomes between the high-dose group and the control group were analyzed. The data showed that different levels of ATZ exposure caused hepatotoxicity in juvenile carp, shown by biochemical parameters and histopathological changes. Comparative transcriptomics showed that high-dose ATZ exposure led to alterations in the expression of various lipid metabolism-related gene changes, including genes associated with metabolic pathways, fatty acid metabolism, and fatty acid elongation. Furthermore, a connection network analysis of the top 100 differentially expressed genes (DEGs) showed a variety of associations between high-dose ATZ-induced liver damage and the principal DEGs, indicating the complexity of hepatotoxicity induced by ATZ. In conclusion, the molecular mechanisms underlying ATZ-triggered hepatotoxicity in juvenile carp are primarily related to impaired lipid metabolism.

Integration of the transcriptome and metabolome reveals the mechanism of resistance to low phosphorus in wild soybean seedling leaves.

Plant growth, development, yield and quality are limited by barren soil. Soil phosphorus deficiency is one of the common factors causing soil barrenness. Plants have evolved morphological, physiological and molecular adaptations to resist to phosphorus deficiency. Wild soybean, a wild relative of cultivated soybean, has an obvious genetic relationship with cultivated soybean and has many beneficial characteristics such as strong low phosphorus resistance. Therefore, in this study, the integration analysis of transcriptome and metabolome of wild and cultivated soybean seedlings leaves were applied under phosphorus deficiency to reveal the mechanism of resistance to low phosphorus stress in wild soybean leaves, especially the key role of membrane phospholipid reuse and protection. Under phosphorus deficiency, wild soybean resisted low phosphorus stress by enhancing phosphorus reuse and strengthening membrane protection mechanisms, that is, by enhancing phospholipid metabolism, degrading membrane phospholipids, releasing phosphorus, increasing phosphorus reuse, and enhancing galactolipid biosynthesis. This, in turn, produced digalactosyl diacylglycerol to replace missing phospholipids for membrane maintenance and enhanced glutathione metabolism to protect the membrane system from damage. At the same time, phosphorus deficiency increased the levels of the intermediate metabolites glycine and ornithine, while significantly regulating the expression of transcription factors WRKY75 and MYB86. The enhancement of these metabolic pathways and the significant regulation of gene expression play an important role in improving the low phosphorus tolerance of wild soybean. This study will provide a useful theoretical basis for breeding soybean with low phosphorus tolerance.

The underlying mechanisms of the effect of superchilling on the tenderness of beef Longissimus lumborum.

This study investigated the effect of superchilling (-30 °C until the core temperature achieved -3 °C, then stored at -1 °C until 24 h, SC) on the tenderness of hot boned beef M. longissimus lumborum (LL), with very fast chilling (-30 °C until the core temperature achieved 0 °C, then stored at -1 °C until 24 h, VFC) and conventional chilling (0- 4 °C for 24 h, CC) as the controls. The lowest initial shear force values were obtained in SC samples compared to those from the VFC and CC treatments (P < 0.05). Clear freezing damage of muscle fibers and more myofibril fragmentation were found in SC samples compared with the other samples early post-mortem. Moreover, SC samples showed the highest level of inosine 5-monophosphate at 3 h post-mortem (P < 0.05). A reduced glycolysis rate (as evidenced by lactate content) was also found in SC treated samples suggesting little contribution of glycolysis on the tenderization of SC.

Boron Oxide B5O6 - Cluster as a Boronyl-Based Inorganic Analog of Phenolate Anion.

Boron oxide clusters have structural richness and exotic chemical bonding. We report a quantum chemical study on the binary B5O6 - cluster, which is relatively oxygen-rich. A global structural search reveals planar C 2v (1A1) geometry as the global minimum structure, featuring a heteroatomic hexagonal B3O3 ring as its core. The three unsaturated B sites are terminated by two boronyl (BO) groups and an O- ligand. The B5O6 - cluster can be faithfully formulated as B3O3(BO)2O-. This structure is in stark contrast to that of its predecessors, C s B5O5 - and T d B5O4 -, both of which have a tetrahedral B center. Thus, there exists a major structural transformation in B5O n - series upon oxidation, indicating intriguing competition between tetrahedral and heterocyclic structures. The chemical bonding analyses show weak 6π aromaticity in the B5O6 - cluster, rendering it a boronyl analog of phenolate anion (C6H5O-) or boronyl boroxine. The calculated vertical detachment energy of B5O6 - cluster is 5.26 eV at PBE0, which greatly surpasses the electron affinities of halogens (Cl: 3.61 eV), suggesting that the cluster belongs to superhalogen anions.

The middle lipin domain adopts a membrane-binding dimeric protein fold.

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

Effects of packaging methods combined with frozen temperature on the color of frozen beef rolls.

Beef rolls for hot pot are usually stored and transported in a frozen state, and the beef color deteriorates quickly. This paper reports on an investigation into the effect of packaging method, freezing temperature and storage time on instrumental color, pH, myoglobin state, lipid oxidation (TBARS) and total volatile basic nitrogen (TVB-N) of beef rolls. It was shown that the color of beef rolls at -18 °C was better than that at -12 °C overall, and the OxyMb% and pH values were higher, while the MetMb% and TBARS were lower with storage at -18 °C. With the extension of storage time, the instrumental color, OxyMb% and pH values of beef rolls decreased. Correspondingly, the MetMb% and TBARS showed an upward trend. However, the TVB-N of all treatments did not exceed the Chinese standard during 180d of storage. The results of this paper provide a number of recommendations for the storage of frozen beef rolls to extend color-shelf life.

A small-scale study on airborne transmission of H9N2 avian influenza virus under field conditions.

INTRODUCTION: H9N2 avian influenza viruses (AIV) can transmit in chicken flocks through direct contact and aerosols. Nevertheless, data on airborne transmission of AIV is very limited, especially under field conditions. To fill this literature gap, this study was designed to investigate airborne transmission of H9N2 AIV originating from infected chicken flocks under field conditions, with the aim to further characterize the airborne transmission of H9N2 AIV. METHODOLOGY: Oropharyngeal swabs were collected from different diseased chickens to confirm H9N2 AIV infection. All glass impingers 30 (AGI-30) were used to collect indoor, upwind and downwind air samples for three chicken houses with H9N2 AIV infected chickens. Swabs and air samples were tested for H9N2 AIV using a real-time reverse transcription polymerase chain reaction (RRT-PCR). H9N2 AIV was isolated in embryonated chicken eggs and hemagglutinin (HA) gene sequence similarity of the isolated AIV was compared. RESULTS: The results showed that indoor air samples were all RRT-PCR positive for H9N2 AIV. Downwind air samples collected between 10 m and 1.5 km away from the chicken houses were also found positive with an average load 2.62-5.21×103 RNA copies/m3. However, upwind air samples were all negative for H9N2 AIV. In addition, H9N2 AIV was isolated from swabs and indoor air samples. CONCLUSION: In summary, this study provides insights into the airborne transmission of H9N2 AIV under field conditions.

The non-homologous end-joining pathway of S. cerevisiae works effectively in G1-phase cells, and religates cognate ends correctly and non-randomly.

DNA double-strand breaks (DSBs) are potentially lethal lesions repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Homologous recombination preferentially reunites cognate broken ends. In contrast, non-homologous end-joining could ligate together any two ends, possibly generating dicentric or acentric fragments, leading to inviability. Here, we characterize the yeast NHEJ pathway in populations of pure G1 phase cells, where there is no possibility of repair using a homolog. We show that in G1 yeast cells, NHEJ is a highly effective repair pathway for gamma-ray induced breaks, even when many breaks are present. Pulsed-field gel analysis showed chromosome karyotypes following NHEJ repair of cells from populations with multiple breaks. The number of reciprocal translocations was surprisingly low, perhaps zero, suggesting that NHEJ preferentially re-ligates the "correct" broken ends instead of randomly-chosen ends. Although we do not know the mechanism, the preferential correct ligation is consistent with the idea that broken ends are continuously held together by protein-protein interactions or by larger scale chromatin structure.

Frizzled-4 C-terminus Distal to KTXXXW Motif is Essential for Normal Dishevelled Recruitment and Norrin-stimulated Activation of Lef/Tcf-dependent Transcriptional Activation.

The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin.

Effect of packaging atmospheres on storage quality characteristics of heavily marbled beef longissimus steaks.

The objective of this study was to investigate the effects of modified atmosphere packaging (MAP) systems on shelf-life and quality of beef steaks with high marbling. Four packaging types were used including 80% O2 MAP (80% O2+20% CO2), 50% O2 MAP (50% O2+30% CO2+20% N2), carbon monoxide MAP (0.4% CO+30% CO2+69.6% N2) and vacuum packaging (VP). Steaks were displayed under simulated retail conditions at 4°C for 12days. Purge loss, pH, color stability, oxidative stability and microbial counts were monitored. Aerobically packaged steaks exhibited a bright-red color at the first 4days. However, discoloration and oxidation became major factors limiting their shelf-life to 8days. Compared with aerobic packaging, anaerobic packaging extended shelf-life of heavily marbled beef steaks, due to better color stability, together with lower oxidation and microbial populations. Among all packaging methods, CO-MAP had the best preservation for steaks, with more red color than other packaging types.