Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis

We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein- containing bands or spots are cut out and inserted directly into a gas- phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150- pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels

The Mr 28,000 gap junction proteins from rat heart and liver are different but related

The sequence of the amino-terminal 32 residues of the rat heart Mr 28,000 gap junction protein presented here allows, for the first time, a sequence comparison of gap junctional proteins from different tissues (heart and liver). Comparison of the rat heart gap junction protein sequence and that available from rat liver reveals 43% sequence identity and conservative changes at an additional 25% of the positions. Both proteins exhibit a hydrophobic domain which could represent a transmembrane span of the junction. This result unequivocally demonstrates the existence of at least two forms of the gap junction protein. As yet, no homology is evident between the gap junctional proteins of either heart or liver and main intrinsic protein from rat eye lens

Developing the buyer-friendly transportation salesperson: an empirical analysis of the most important seller traits and behaviors from the transportation buyer’s perspective

The research reported in this manuscript provides several insights regarding the specific behaviors and traits of transportation salespersons as sought by a sample of shippers. Shippers in both manufacturing and non-manufacturing industries were asked to evaluate the importance of 30 potential salesperson characteristics. Overall, dependability, ethical conduct, honesty, provision of regular service, and solution selling were ranked as the most important (“must have”) characteristics. In addition to the overall rankings, t-tests were used to compare the manufacturing and non-manufacturing groups and ANOVA tests were used to compare the responses of shippers which were grouped by number of contacts from salespersons

Truckload transportation requirements: in anticipation of Y2K with epilogue

This article investigates the impact of the much-hyped Y2K phenomenon on truckload transportation requirements in the United States, as a result of year-end inventory build-ups. The article reports the results of a Y2K Truckload Transportation Survey of truckload shippers conducted in August of 1999. Additionally, the article takes a post-hoc look at what actually occurred in an effort to completely document the impact of the Y2K phenomenon in the dry van, temperature controlled, and flatbed segments of the truckload transportation industry

Identification of a novel retroviral gene unique to human immunodeficiency virus type 2 and simian immunodeficiency virus SIVMAC

Human and simian immunodeficiency-associated retroviruses are extraordinarily complex, containing at least five genes, tat, art, sor, R, and 3' orf, in addition to the structural genes gag, pol, and env. Recently, nucleotide sequence analysis of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus SIVMAC revealed the existence of still another open reading frame, termed X, which is highly conserved between these two viruses but absent from HIV-1. In this report, we demonstrate for the first time that the X open reading frame represents a functional retroviral gene in both HIV-2 and SIVMAC and that it encodes a virion-associated protein of 14 and 12 kilodaltons, respectively. We also describe the production of recombinant TrpE/X fusion proteins in Escherichia coli and show that sera from some HIV-2-infected individuals specifically recognize these proteins

Template-Directed Ligation of Peptides to Oligonucleotides

Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template

A new approach toward PTP-1B inhibition

Abstract only availableSignaling pathways for cellular metabolism, growth, proliferation, differentiation, immune response, motility, and tissue homeostasis is regulated by the phosphorylation of protein tyrosine residues on target proteins in the relevant signal transduction pathways. Phosphorylation levels of tyrosine residues are controlled by the opposing actions of two enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Protein tyrosine kinases add phosphoryl groups while PTPs catalyze their removal. PTPs are emerging as potential drug targets for the treatment of type 2 diabetes, autoimmune diseases, osteoporosis, and cancer. PTP-1B is the archetypal PTP and its inactivation may be a viable treatment for type 2 diabetes and obesity. PTP-1B is regulated by endogenous hydrogen peroxide (H2O2), which is a known cellular signaling agent. H2O2 oxidatively-inactivates PTP-1B, and its activity is regenerated by free thiols within the cell such as glutathione. In order to facilitate enzyme regeneration the aforementioned thiol must have access to the enzyme active site. We are testing the hypothesis that small molecules can inhibit thiol-mediated reactivation of redox-inactivated PTPs. Molecules with such a property would decrease the activity of target PTPs in cells, thus enhancing cellular response to external stimuli that act through receptor protein kinases.Life Sciences Undergraduate Research Opportunity Progra