Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Phytochrome controlled acid RNase: An “attached” protein of ribosomes

The light-dependent increment in RNase activity (which is ribosome bound in cell extracts) is distributed as a gradient increasing from base to hook of lupin hypocotyls. No evidence was found of non-specific or of specific activation of pre-formed enzyme molecules following isolation, either before or after (latent activity) destruction of particles. The autodegradation capacity of ribosomes isolated from irradiated cells was almost double that of ribosomes from etiolated tissue. It is proposed that association between the bulk of the light-controlled RNase fraction and lupin ribosomes results from binding of soluble protein. It is not clear whether binding is specific or an artifact of isolation.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

A new tubulin-binding protein

A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indiscriminate polymerization, or to promote ordered nucleation by maintaining high local concentrations.     

Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

Biochemistry and genetics of taste- and odor-producing cyanobacteria

Cyanobacteria are one of the principal sources of volatile organic compounds (VOCs) which cause offensive taste and odor (T&O) in drinking and recreational water, fish, shellfish and other seafood. Although non-toxic to humans, these T&O compounds severely undermine public trust in these commodities, resulting in substantial costs in treatment, and lost revenue to drinking water, aquaculture, food and beverage and tourist/hospitality industries. Mitigation and control have been hindered by the complexity of the communities and processes which produce and modify T&O events, making it difficult to source-track the major producer(s) and the factors governing VOC production and fate. Over the past decade, however, advances in bioinformatics, enzymology, and applied detection technologies have greatly enhanced our understanding of the pathways, the enzymes and the genetic coding for some of the most problematic VOCs produced by cyanobacteria. This has led to the development of tools for rapid and sensitive detection and monitoring for the VOC production at source, and provided the basis for further diagnostics of endogenous and exogenous controls. This review provides an overview of current knowledge of the major cyanobacterial VOCs, the producers, the biochemistry and the genetics and highlight the current applications and further research needs in this area.     

严重急性呼吸综合征冠状病毒2膜蛋白对宿主细胞pre-mRNA 3"UTR加工的影响

目的 研究严重急性呼吸综合征冠状病毒2（SARS-CoV-2）膜蛋白对宿主细胞mRNA前体（pre-mRNA）3"非翻译区（UTR）加工的影响。方法 本研究以人肺上皮细胞系A549为模型，利用瞬时转染在细胞内过表达SARS-CoV-2膜蛋白；利用RNA-Seq测序技术及生物信息学分析方法，系统性描绘宿主细胞选择性多聚腺苷酸化（alternative polyadenylation，APA）事件；Metascape数据库对发生显著APA变化的基因进行功能富集分析；RT-qPCR验证靶基因3"UTR长度变化；蛋白质免疫印迹（Western blot）检测目的蛋白表达水平。结果 SARS-CoV-2膜蛋白外源表达后宿主细胞内共813个基因发生显著APA变化。GO和KEGG分析显示，差异APA基因广泛参与有丝分裂细胞周期、调节细胞应激等生物过程，涉及病毒感染和蛋白质加工等。从中进一步筛选出AKT1基因，在IGV软件中显示3"UTR延长；RT-qPCR验证AKT1基因的3"UTR长度变化趋势；Western blot结果显示AKT1蛋白磷酸化水平增加。结论 SARS-CoV-2膜蛋白潜在影响宿主pre-mRNA的3"UTR加工，其中参与多种病毒性生物过程的AKT1基因 3"UTR延长，且其编码的蛋白质功能在细胞内被激活。     

Synthesis of an Asymmetrically Substituted AZA Crown Ether as Metal and Amino Acid Binding Site in DNA Conjugates

Abstract

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.