Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up-regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non-muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10-ethylenetetrahydrofolated reductase (5,10-MTHFR), angiotensin-converting enzyme 2 (ACE2), co-lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI-3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.     

Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle

Skeletal muscle phosphorylase kinase (PhK) is a Ca²⁺-dependent enzyme complex, (αβγδ)₄, with the δ subunit being tightly bound endogenous calmodulin (CaM). The Ca²⁺-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the “excitation-contraction-energy production triad.” Although the Ca²⁺-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic γ and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the γ subunit sharing high sequence similarity with the Ca²⁺-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca²⁺ stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca²⁺ of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca²⁺-binding proteins troponin C and CaM.     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.     

RAPTURE (RAD capture) panel facilitates analyses characterizing sea lamprey reproductive ecology and movement dynamics

Genomic tools are lacking for invasive and native populations of sea lamprey (Petromyzon marinus). Our objective was to discover single nucleotide polymorphism (SNP) loci to conduct pedigree analyses to quantify reproductive contributions of adult sea lampreys and dispersion of sibling larval sea lampreys of different ages in Great Lakes tributaries. Additional applications of data were explored using additional geographically expansive samples. We used restriction site‐associated DNA sequencing (RAD‐Seq) to discover genetic variation in Duffins Creek (DC), Ontario, Canada, and the St. Clair River (SCR), Michigan, USA. We subsequently developed RAD capture baits to genotype 3,446 RAD loci that contained 11,970 SNPs. Based on RAD capture assays, estimates of variance in SNP allele frequency among five Great Lakes tributary populations (mean F_ST 0.008; range 0.00–0.018) were concordant with previous microsatellite‐based studies; however, outlier loci were identified that contributed substantially to spatial population genetic structure. At finer scales within streams, simulations indicated that accuracy in genetic pedigree reconstruction was high when 200 or 500 independent loci were used, even in situations of high spawner abundance (e.g., 1,000 adults). Based on empirical collections of larval sea lamprey genotypes, we found that age‐1 and age‐2 families of full and half‐siblings were widely but nonrandomly distributed within stream reaches sampled. Using the genomic scale set of SNP loci developed in this study, biologists can rapidly genotype sea lamprey in non‐native and native ranges to investigate questions pertaining to population structuring and reproductive ecology at previously unattainable scales.     

Detailed N-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognises both mannosylated and sulphated ligands through its C-type lectin domains (CTLDs) and cysteine-rich (CR) domain, respectively. It is widely expressed among different tissues and by certain cell types in vivo. Our previous study suggested that the glycosylation, especially terminal sialylation, regulated the functional specificities of MR. In the current investigation, the distribution of MR among various mouse tissues was studied and the N-linked glycosylation of spleen MR was analysed. Our results showed that spleen expressed the most abundant MR, consistent with its wide distribution in different cell types in this organ. Spleen MR was heterogeneously N-glycosylated. The majority of the glycans were sialylated in the α2 → 6-linkage and both Neu5Ac and Neu5Gc sialic acids were detected. Most glycans were bi-antennary (74%) with ∼22% tri-antennary and most were core fucosylated (68%). About 13% contained α-galactose. In the lung, MR exhibited more terminal sialic acids in the α2 → 3- rather than in the α2 → 6-configuration. Our study provides a profile of MR N-linked glycosylation that will facilitate our understanding of their physiological role on MR biology in vivo.     

Catalytic Turnover-Based Phage Selection for Engineering the Substrate Specificity of Sfp Phosphopantetheinyl Transferase

We report a high-throughput phage selection method to identify mutants of Sfp phosphopantetheinyl transferase with altered substrate specificities from a large library of the Sfp enzyme. In this method, Sfp and its peptide substrates are co-displayed on the M13 phage surface as fusions to the phage capsid protein pIII. Phage-displayed Sfp mutants that are active with biotin-conjugated coenzyme A (CoA) analogues would covalently transfer biotin to the peptide substrates anchored on the same phage particle. Affinity selection for biotin-labeled phages would enrich Sfp mutants that recognize CoA analogues for carrier protein modification. We used this method to successfully change the substrate specificity of Sfp and identified mutant enzymes with more than 300-fold increase in catalytic efficiency with 3′-dephospho CoA as the substrate. The method we developed in this study provides a useful platform to display enzymes and their peptide substrates on the phage surface and directly couples phage selection with enzyme catalysis. We envision this method to be applied to engineering the catalytic activities of other protein posttranslational modification enzymes.     

siRNA release from gold nanoparticles by nanosecond pulsed laser irradiation and analysis of the involved temperature increase

Nanosecond pulsed laser irradiation can trigger a release of nucleic acids from gold nanoparticles, but the involved nanoeffects are not fully understood yet. Here we investigate the release of coumarin labeled siRNA from 15 to 30 nm gold particles after nanosecond pulsed laser irradiation. Temperatures in the particle and near the surface were calculated for the different radiant exposures. Upon irradiation with laser pulses of 4 nanosecond duration release started for both particle sizes at a calculated temperature increase of approximately 500 K. Maximum coumarin release was observed for 15 nm particles after irradiation with radiant exposure of 80 mJ cm^?2 and with 32 mJ cm^?2 for 30 nm particles. This corresponds to a temperature increase of 815 and 900 K, respectively. Our results show that the molecular release by nanosecond pulsed irradiation is based on a different mechanism compared to continuous or femtosecond irradiation. Local temperatures are considerably higher and it is expected that bubble formation plays a crucial role in release and damage to cellular structures.      

Effects of weak laser radiation (632.8 nm) on isolated mouse immune cells

The dose dependence of in vitro effects of low-intensity radiation of a He-Ne laser (632.8 nm, 0.2 mW/cm²) on the functional activity of peritoneal macrophages and lymphocytes of mouse spleen was studied. The exposure of isolated cells was varied from 5 to 180 s. If the exposure did not exceed 60 s, stimulation of secretory activity was observed: increased production of interleukin 2, interferon γ, and interleukin 6 in lymphocytes; increased production of tumor necrosis factor α, nitric oxide, and interleukin 6 in macrophages; and enhanced activity of natural killer cells. A longer exposure (up to 180 s) either had no effect on the synthesis of certain cytokines (interleukin 2 in lymphocytes and interleukin 6 in macrophages) or inhibited it, which was expressed in decreased production of interleukin 6 and interferon γ in lymphocytes and nitric oxide in macrophages, as well as in suppression of the activity of natural killer cells. Conversely, the production of interleukin 3 decreased after a short-term exposure but increased after 180-s irradiation. The high sensitivity of cells to extremely weak laser light also manifested itself as a considerable increase in expression of the inducible heat shock protein 70; this effect was observed at all doses studied, including the 5-s exposure. In contrast, expression of the heat shock protein 90 slightly decreased after irradiation of cells with laser light.     

d-Ribose glycates β2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β₂-Microglobulin (β₂M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β₂M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β₂M in vitro. We find that the glycating agent d-ribose interacts with human β₂M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β₂M molecules are highly oligomerized compared with unglycated β₂M, and have granular morphology. Furthermore, such ribosylated β₂M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0 mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β₂M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β₂M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β₂M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β₂M-associated diseases such as HAA.