Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

Regulation of neurocoel morphogenesis by Pard6 gamma b

The Par3/Par6/aPKC protein complex plays a key role in the establishment and maintenance of apicobasal polarity, a cellular characteristic essential for tissue and organ morphogenesis, differentiation and homeostasis. During a forward genetic screen for liver and pancreas mutants, we identified a pard6γb mutant, representing the first known pard6 mutant in a vertebrate organism. pard6γb mutants exhibit defects in epithelial tissue development as well as multiple lumens in the neural tube. Analyses of the cells lining the neural tube cavity, or neurocoel, in wildtype and pard6γb mutant embryos show that lack of Pard6γb function leads to defects in mitotic spindle orientation during neurulation. We also found that the PB1 (aPKC-binding) and CRIB (Cdc-42-binding) domains and the KPLG amino acid sequence within the PDZ domain (Pals1-and Crumbs binding) are not required for Pard6γb localization but are essential for its function in neurocoel morphogenesis. Apical membranes are reduced, but not completely absent, in mutants lacking the zygotic, or both the maternal and zygotic, function of pard6γb, leading us to examine the localization and function of the three additional zebrafish Pard6 proteins. We found that Pard6α, but not Pard6β or Pard6γa, could partially rescue the pard6γb^s441 mutant phenotypes. Altogether, these data indicate a previously unappreciated functional diversity and complexity within the vertebrate pard6 gene family.     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney

The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N-linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina.     

Usefulness of sugar mapping by liquid chromatography/mass spectrometry in comparability assessments of glycoprotein products.

We have previously reported that sugar-mapping by liquid chromatography/mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) can be useful for structural analysis of carbohydrates in a glycoprotein. In this paper, we evaluated sugar-mapping with regard to its use in comparability assessment of glycoprotein products. Erythropoietins (EPO) produced from three different sources were chosen as models of the closely related glycoprotein products. The two-dimensional displays of sugar maps drawn by LC/MS with GCC clearly showed the differences in carbohydrate heterogeneity with regard to sialylation, acetylation, and sulphation patterns among three EPOs. Exoglycosidase digestion followed by sugar-mapping provided information regarding the structure of characteristic carbohydrates in each EPO. These results demonstrate that LC/MS with GCC can reveal the details of carbohydrate heterogeneity in order to distinguish between closely related glycoprotein products. Our method can thus be useful in comparability assessments of therapeutic glycoproteins.     

Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage

Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1^Ser137 and pPlk1^Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1^Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1^Thr210 was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1^Ser137 was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1^Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1^Ser137 accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1^Ser137 and pPlk1^Thr210, as well as the subcellular distribution of pPlk1^Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1^Ser137 is the action executor, in mouse oocytes during meiotic division.     

A method for fast safety screening of explosives in terms of crystal packing and molecular stability

Safety prediction is crucial to the molecular design or the material design of explosives, and the predictions based on any single factor alone will cause much inaccuracy, leading to a desire for a method on multi-bases. The presented proposes an improved method for fast screening explosive safety by combining a crystal packing factor and a molecular one, that is, steric hindrance against shear slide in crystal and molecular stability, denoted by intermolecular friction symbol (IFS) and bond dissociation energy (BDE) of trigger linkage respectively. Employing this BDE-IFS combined method, we understand the impact sensitivities of 24 existing explosives, and predict those of two energetic-energetic cocrystals of the observed CL-20/BTF and the supposed HMX/TATB. As a result, a better understanding is implemented by the combined method relative to molecular stability alone, verifying its improvement of more accurate predictions and the feasibility of IFS to graphically reflect molecular stacking in crystals. Also, this work verifies that the explosive safety is strongly related with its crystal stacking, which determines steric hindrance and influences shear slide.     

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency

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Genetic mapping identifies a rice naringenin O-glucosyltransferase that influences insect resistance

Naringenin, the biochemical precursor for predominant flavonoids in grasses, provides protection against UV damage, pathogen infection and insect feeding. To identify previously unknown loci influencing naringenin accumulation in rice (Oryza sativa), recombinant inbred lines derived from the Nipponbare and IR64 cultivars were used to map a quantitative trait locus (QTL) for naringenin abundance to a region of 50 genes on rice chromosome 7. Examination of candidate genes in the QTL confidence interval identified four predicted uridine diphosphate-dependent glucosyltransferases (Os07g31960, Os07g32010, Os07g32020 and Os07g32060). In vitro assays demonstrated that one of these genes, Os07g32020 (UGT707A3), encodes a glucosyltransferase that converts naringenin and uridine diphosphate-glucose to naringenin-7-O-β-d -glucoside. The function of Os07g32020 was verified with CRISPR/Cas9 mutant lines, which accumulated more naringenin and less naringenin-7-O-β-d -glucoside and apigenin-7-O-β-d -glucoside than wild-type Nipponbare. Expression of Os12g13800, which encodes a naringenin 7-O-methyltransferase that produces sakuranetin, was elevated in the mutant lines after treatment with methyl jasmonate and insect pests, Spodoptera litura (cotton leafworm), Oxya hyla intricata (rice grasshopper) and Nilaparvata lugens (brown planthopper), leading to a higher accumulation of sakuranetin. Feeding damage from O. hyla intricata and N. lugens was reduced on the Os07g32020 mutant lines relative to Nipponbare. Modification of the Os07g32020 gene could be used to increase the production of naringenin and sakuranetin rice flavonoids in a more targeted manner. These findings may open up new opportunities for selective breeding of this important rice metabolic trait.     

Bag3-Induced Autophagy Is Associated with Degradation of JCV Oncoprotein, T-Ag

JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag), in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag) family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases.