Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

环氧树脂固定化的Bacillus sp.DL-2胞外蛋白酶在拆分(±)-乙酸苏合香酯中的应用

环氧基是一个非常活跃的基团,它能与酶、蛋白质和核酸等生物分子发生反应形成共价键,有利于生物分子的固定化。经共价结合法固定化的酶其稳定性及重复使用性可得到显著提高。用环氧树脂ES-103B为载体采用共价结合法对海洋细菌Bacillus sp. DL-2的胞外蛋白酶进行固定化,经过单因素实验优化条件得出最优固定化条件为:p H 8. 0的胞外蛋白酶溶液,25 g/L的ES-103B,45℃下反应8h。采用此最优条件下的固定化酶拆分(±)-乙酸苏合香酯制备出了e. e. p=97. 5%的(R)-1-苯乙醇(产率为45. 0%)和e. e. s=99. 2%的(S)-乙酸苏合香酯(产率为83. 9%)。该固定化酶拆分(±)-乙酸苏合香酯在重复使用8次后制备出的(R)-1-苯乙醇的e. e. p仍大于90%,且固定化胞外蛋白酶在4℃下具有较好的储存稳定性。     

A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.     

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.     

Structural Decomposition Analysis of Raw Material Consumption

The aim of this article is to quantify the drivers for the changes in raw material consumption (domestic material consumption expressed in the form of all materials extracted and used in the production phase) in terms of technology, which refers to the concept of sustainable production; the product structure of final demand, which refers to the concept of sustainable consumption; and the volume of final demand, which is related to economic growth. We also aim to determine to what extent the technological development and a shift in product structure of the final demand compensate for the growth in final consumption volume. Therefore, we apply structural decomposition analysis (SDA) to the change in raw material consumption (RMC) of the Czech Republic between 2000 and 2007. To present the study in a broader context, we also show other material flow indicators for the Czech Republic for 2000 and 2007. Our findings of SDA show that final demand structure has a very limited effect on the change in material flows. The rapid change in final demand volume was not compensated for crude oil, metal ores, construction materials, food crops, and timber. For the material category of non‐iron metal ores, even the change in technology contributes to an increase in material flows. The largest relative increases are reported for non‐iron metal ores (38%) and construction materials (30%). The main changes in material flows related to the Czech Republic are driven by exports and enabled by imports, the main source of these increased material flows. This emphasizes the increasing role of international trade.     

Solubility characteristics of Micrococcus lysodeikticus membrane components in detergents and chaotropic salts analyzed by immunoelectrophoresis

In order to evaluate the effectiveness and selectivity of various reagents in the solubilization of bacterial membranes, membranes of Micrococcus lysodeikticus were treated with detergents and chaotropic agents. The composition of the extracts so obtained was analyzed by rocket and two-dimensional immunoelectrophoretic techniques. Recovery of succinate-, malate-, and reduced nicotinamide adenine dinucleotide- (NADH) dehydrogenases, ATPase, succinylated lipomannan and cytochromes in the extracts was measured. Treatment with a variety of non-denaturing detergents produced extracts that were generally qualitatively uniform although quantitative differences were observed. The degree of extraction of various components was correlated with the hydrophile-lipophile balance. Several chaotropic agents were also evaluated as reagents for membrane solubilization. These agents were less effective in extraction of bulk protein, but produced extracts enriched in some membrane components.     

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.     

Grouped sequential exploitation of food patches in a flock feeder,the feral pigeon

Feral and laboratory flocks of rock doves ($\text{Columba}$$\text{livia}$) show a pattern of grouped sequential exploitation when simultaneously presented with two dispersed, depleting patches of seed. This behavior contrasts with the ideal free distribution pattern shown when patches are small and concentrated. Grouped sequential exploitation consists of two phases: all pigeons first land together and feed at one patch, then leave one by one for the other patch. Departure times of individuals for the second patch are correlated with feeding rate at patch 1, which is in turn correlated with position in the dominance hierarchy. The decision to switch from patch 1 to patch 2 improves individual feeding rates in all cases, but is done slightly later than it should according to optimal foraging theory.     

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.