Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.     

Structural stability and reversible unfolding of recombinant porcine S100A12

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca2+ homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn2+ or Ca2+ with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses.     

Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.     

S100A14, a Member of the EF-hand Calcium-binding Proteins,Is Overexpressed in Breast Cancer and Acts as a Modulator of HER2 Signaling

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK. S100A14, one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens, and there is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding. Moreover, we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.     

Proteins of the Brain Extracellular Fluid: Evidence for Release of S-100 Protein

Abstract: Extracellular protein fractions were obtained (1) by mild, isotonic irrigation of freshly perfused brain tissue; (2) by collection of proteins released into super-fusing medium by physiologically viable slices of rat hippocampus; and (3) by sampling the CSF of anesthetized rats. Analysis of the S-100 protein content of these fractions gave values of 2.8, 4.2, and 1.8 μg S-100/mg protein, respectively. These values were three- to sixfold higher than the S-100 content of the soluble cytoplasmic protein fractions from the same tissue. This several-fold higher S-100 content of the extracellular protein fractions relative to the intracellular cytoplasmic protein fractions indicates that S-100 is selectively released into the extracellular spaces of the brain. We suggest that the biological function of this CNS protein may involve intercellular transfer.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

Functional properties of the newly observed γ-chain fetal hemoglobin variant Hb F-Monserrato-Sassari (HBG2:c.280T > C) or [γ93 (F9) Cys → Arg]

Background

HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population.

Methods

Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined.

Results

Sequencing of the γ globin genes revealed the TGT → CGT transition at codon 93 in one of the two ^Gγ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position β93 are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity.

General significance

Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T → R transition of the HbF tetramer. Substitutions at the F9 position of the ^Gγ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport.     

青藏高原东缘常见种长毛风毛菊(Saussurea hieracioides)的繁殖分配

对分布在青藏高原东缘高寒草甸常见种长毛风毛菊的6个海拔梯度(3200～3850m)18个居群的繁殖分配进行了研究,结果表明:长毛风毛菊随海拔的升高个体和营养器官减小,繁殖的投入增加;随着海拔的升高,长毛风毛菊的个体管状小花数目和种子数目减少,管状小花重量和百粒重增大;个体管状小花的数目及重量和个体种子的数目及重量间存在权衡关系;长毛风毛菊的个体大小和繁殖分配存在负相关关系。在资源受限的环境下,长毛风毛菊以减少管状小花数目和种子的数目来增加管状小花重量和百粒重,是保证有性繁殖成功的策略之一。     

Controlled release of d-glucose from starch granules containing 29% free d-glucose and Eudragit L100-55 as a binding and coating agent

Waxy maize starch (100% amylopectin) granules were modified by reaction of the granules with glucoamylase in a minimum amount of water to give 29% (w/w) d-glucose inside the granules [Kim, Y.-K.; Robyt, J. F. Carbohydr. Res.1999, 318, 129−134]. These granules were made into beads by dropping an ethanol slurry of starch and different amounts of Eudragit L100-55 in a constant ratio of 100:1 from a pipette onto Whatman 3MM filter paper. The starch beads were air dried and then repeatedly sprayed 0-12 times with 2.0% (w/v) Eudragit L100-55 in ethanol, with drying between each spraying, to coat the surface of the starch beads, giving different amounts of Eudragit L100-55 coating. Seven different kinds of beads, with different amounts of Eudragit L100-55 binding and coating agent, were obtained. The rates of release of d-glucose into water from the seven kinds of beads were inversely proportional to the amount of binding and coating agent. Bead type I, which was without any binding and coating gave a fast 100% release of d-glucose in 30 min. Beads II and III also gave a fast 100% release in 60 min and 90 min, respectively. Bead IV gave a near linear release of 97% d-glucose in 150 min; Bead V gave a 50% release in 120 min followed by the remaining 50% in 60 min; and Beads VI and VII gave a slow release of 10% and 4%, respectively, from 0 to 120 min, followed by a rapid 100% release from 120 to 180 min.