Snapin, a new regulator of receptor signaling, augments alpha1A-adrenoceptor-operated calcium influx through TRPC6

Activation of G(q)-protein-coupled receptors, including the alpha(1A)-adrenoceptor (alpha(1A)-AR), causes a sustained Ca(2+) influx via receptor-operated Ca(2+) (ROC) channels, following the transient release of intracellular Ca(2+). Transient receptor potential canonical (TRPC) channel is one of the candidate proteins constituting the ROC channels, but the precise mechanism linking receptor activation to increased influx of Ca(2+) via TRPCs is not yet fully understood. We identified Snapin as a protein interacting with the C terminus of the alpha(1A)-AR. In receptor-expressing PC12 cells, co-transfection of Snapin augmented alpha(1A)-AR-stimulated sustained increases in intracellular Ca(2+) ([Ca(2+)](i)) via ROC channels. By altering the Snapin binding C-terminal domain of the alpha(1A)-AR or by reducing cellular Snapin with short interfering RNA, the sustained increase in [Ca(2+)](i) in Snapin-alpha(1A)-AR co-expressing PC12 cells was attenuated. Snapin co-immunoprecipitated with TRPC6 and alpha(1A)-AR, and these interactions were augmented upon alpha(1A)-AR activation, increasing the recruitment of TRPC6 to the cell surface. Our data suggest a new receptor-operated signaling mechanism where Snapin links the alpha(1A)-AR to TRPC6, augmenting Ca(2+) influx via ROC channels.     

A role for Lte1p (a low temperature essential protein involved in mitosis) in proprotein processing in the yeast secretory pathway

We previously identified six single gene disruptions in Saccharomyces cerevisiae that allow enhanced immunoreactive insulin secretion primarily because of defective Kex2p-mediated endoproteolytic processing. Five eis mutants disrupted established VPS (vacuolar protein sorting) genes, The sixth, LTE1, is a Low Temperature (<15 degrees C) Essential gene encoding a large protein with potential guanine nucleotide exchange (GEF) domains. Lte1p functions as a positive regulator of the mitotic GTPase Tem1p, and overexpression of Tem1p suppresses the low temperature mitotic defect of lte1. By sequence analysis, Tem1p has highest similarity to Vps21p (yeast homolog of mammalian Rab5). Unlike TEM1, LTE1 is not restricted to mitosis but is expressed throughout the cell cycle. Lte1p function in interphase cells is largely unknown. Here we confirm the eis phenotype of lte1 mutant cells and demonstrate a defect in proalpha factor processing that is rescued by expression of full-length Lte1p but not a C-terminally truncated Lte1p lacking its GEF homology domain. Neither overexpression of Tem1p nor 13 other structurally related GTPases can suppress the secretory proprotein processing defect. However, overexpression of Vps21p selectively restores proprotein processing in a manner dependent upon the active GTP-bound form of the GTPase. By contrast, a vps21 mutant produces a synthetic defect with lte1 in proprotein processing, as well as a synthetic growth defect. Together, the data underscore a link between the mitotic regulator, Lte1p, and protein processing and trafficking in the secretory/endosomal system.     

Single protein production (SPP) system in Escherichia coli

Here, we provide a detailed protocol for the single protein production (SPP) system, which is designed to produce only a single protein of interest in living Escherichia coli cells. Induction of MazF, an mRNA interferase that cleaves RNA at ACA nucleotide sequences, results in complete cell growth arrest. However, if mRNA encoding a protein of interest is engineered to be devoid of ACA base triplets and is induced at 15 degrees C using pCold vectors in MazF-expressing cells, only the protein from this mRNA is produced at a yield of 20-30% of total cellular protein; other cellular protein synthesis is almost completely absent. In theory, any protein can be produced by the SPP system. Protein yields are typically unaffected even if the culture is condensed up to 40-fold, reducing the cost of protein production by up to 97.5%. The SPP system has a number of key features important for protein production, including high-yield and prolonged production of isotope-labeled protein at a very high signal-to-noise ratio. The procedure can be completed in 7 d after cloning of an ACA-less target gene into the expression system.     

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L⁻¹ 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA₃) at 1.0 μmol L⁻¹ stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L⁻¹ of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.     

Helicobacter pylori dampens gut epithelial self-renewal by inhibiting apoptosis, a bacterial strategy to enhance colonization of the stomach

Colonization of the gastric pits in the stomach by Helicobacter pylori (Hp) is a major risk factor for gastritis, gastric ulcers, and cancer. Normally, rapid self-renewal of gut epithelia, which occurs by a balance of progenitor proliferation and pit cell apoptosis, serves as a host defense mechanism to limit bacterial colonization. To investigate how Hp overcomes this host defense, we use the Mongolian gerbil model of Hp infection. Apoptotic loss of pit cells induced by a proapoptotic agent is suppressed by Hp. The ability of Hp to suppress apoptosis contributed to pit hyperplasia and persistent bacterial colonization of the stomach. Infection with WT Hp but not with a mutant in the virulence effector cagA increased levels of the prosurvival factor phospho-ERK and antiapoptotic protein MCL1 in the gastric pits. Thus, CagA activates host cell survival and antiapoptotic pathways to overcome self-renewal of the gastric epithelium and help sustain Hp infection.     

Lipopolysaccharide enhances the production of nicotine-induced prostaglandin E2 by an increase in cyclooxygenase-2 expression in osteoblasts

Previous studies have indicated that lipopolysaccharide(LPS)from Gram-negative bacteria inplaque induces the release of prostaglandin E_2(PGE_2),which promotes alveolar bone resorption in periodontitis,and that tobacco smoking might be an important risk factor for the development and severity of periodontitis.We determined the effect of nicotine and LPS on alkaline phosphatase(ALPase)activity,PGE_2 production,and the expression of cyclooxygenase(COX-1,COX-2),PGE_2 receptors Ep1-4,and macrophage colonystimulating factor(M-CSF)in human osteoblastic Saos-2 cells.The cells were cultured with 10~(-3)M nicotinein the presence of 0,1,or 10μg/ml LPS,or with LPS alone.ALPase activity decreased in cells cultured withnicotine or LPS alone,and decreased further in those cultured with both nicotine and LPS,whereas PGE_2production significantly increased in the former and increased further in the latter.By itself,nicotine did notaffect expression of COX-1,COX-2,any of the PGE_2 receptors,or M-CSF,but when both nicotine and LPSwere present,expression of COX-2,Ep3,Ep4,and M-CSF increased significantly.Simultaneous addition of10~(-4)M indomethacin eliminated the effects of nicotine and LPS on ALPase activity,PGE_2 production,and M-CSF expression.Phosphorylation of protein kinase A was high in cells cultured with nicotine and LPS.Theseresults suggest that LPS enhances the production of nicotine-induced PGE_2 by an increase in COX-2 expres-sion in osteoblasts,that nicotine-LPS-induced PGE_2 interacts with the osteoblast Ep4 receptor primarily inautocrine or paracrine mode,and that the nicotine-LPS-induced PGE_2 then decreases ALPase activity andincreases M-CSF expression.     

Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.