Isolation and identification of a peptide and its cDNA from the mosquito Aedes aegypti related to Manduca sexta allatotropin

Immunocytochemistry revealed that an allatotropin-immunoreactive peptide is produced by several neuroendocrince cells in the abdominal ganglia of the mosquito Aedes aegypti. The immunoreactive peptide was isolated and its structure determined to be Ala-Pro-Phe-Arg-Asn-Ser-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-amide. A cDNA clone encoding this novel neuropeptide was shown to encode a single copy of this peptide. The cDNA is unusual in that the first seven ATGs are not used for translation initiation.     

Electrospray ionization mass spectrometry in the study of biomolecular non-covalent interactions

In the past mass spectrometry has been limited to the study of small, stable molecules, however, with the emergence of electrospray ionization mass spectrometry (ESI-MS) large biomolecules as well as non-covalent biomolecular complexes can be studied. ESI-MS has been used to study non-covalent interactions involving proteins with metals, ligands, peptides, oligonucleotides, as well as other proteins. Although complementary to other well-established techniques such as circular dichroism and fluorescence spectroscopy, ESI-MS offers some advantages in speed, sensitivity, and directness particularly in the determination of the stoichiometry of the complex. One major advantage is the ability of ESI-MS to provide multiple signals each arising from a distinct population within the sample. In this review I will discuss some of the different types of non-covalent biomolecular interactions that have been studied using ESI-MS, highlighting examples which show the efficacy of using ESI-MS to probe the structure of biomolecular complexes.     

A diverse family of inositol 5-phosphatases playing a role in growth and development in Dictyostelium discoideum

Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with a SacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain.     

Characterization of the low molecular weight human serum proteome

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.     

Multivessel revascularisation versus infarct-related artery only revascularisation during the index primary PCI in STEMI patients with multivessel disease: a meta-analysis

Background

There are controversial data regarding infarct-related artery only (IRA-PCI) revascularisation versus multivessel revascularisation (MV-PCI) in ST-elevation myocardial infarction (STEMI) patients with multivessel disease undergoing primary percutaneous coronary intervention (PCI). We performed a meta-analysis comparing outcome in same stage MV-PCI versus IRA-PCI in STEMI patients with multivessel disease.

Methods

Systematic searches of studies comparing MV-PCI with IRA-PCI in the MEDLINE and the Cochrane Database of systematic reviews were conducted. A meta-analysis was performed of all available studies. Primary outcome was all-cause mortality. Secondary endpoints were re-infarction, revascularisation, bleeding and major adverse cardiac events (MACE).

Results

A total of 15 studies were identified with a total number of 35,975 patients. Mortality rate was significantly higher in the MV-PCI group compared with the IRA-PCI group, odds ratio (OR): 1.64 (1.46–1.85). Both the incidence of re-infarction and re-PCI were significantly lower in the MV-PCI group compared with the IRA-PCI group: OR 0.54 (0.34–0.88) and OR 0.67 (0.48–0.93), respectively. Bleeding complications occurred more often in the MV-PCI group as compared with the IRA-PCI group: OR 1.24 (1.08–1.42). Rates of MACE were comparable between the two groups.

Conclusions

MV-PCI during the index of primary PCI in STEMI patients is associated with a higher mortality rate, a higher risk of bleeding complications, but lower risk of re-intervention and re-infarction and comparable rates of MACE.     

Macrocyclic BACE-1 inhibitors acutely reduce Aβ in brain after po application

A series of macrocyclic peptidic BACE-1 inhibitors was designed. While potency on BACE-1 was rather high, the first set of compounds showed poor brain permeation and high efflux in the MDRI–MDCK assay. The replacement of the secondary benzylamino group with a phenylcyclopropylamino group maintained potency on BACE-1, while P-glycoprotein-mediated efflux was significantly reduced and brain permeation improved. Several compounds from this series demonstrated acute reduction of Aβ in human APP-wildtype transgenic (APP51/16) mice after oral administration.     

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.     

Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin

Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of ezrin and moesin, the two ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.