A Review of the Salivary Proteome and Peptidome and Saliva-derived Peptide Therapeutics

Saliva is a glandular secretion that is vital in the maintenance of healthy oral tissues. In this review we outline the high abundance salivary proteins, summarise the status of the salivary proteome and peptidome, the genetic origin and recognised functions of these proteins, the diseases associated with salivary disorders, and the emerging saliva-derived peptide therapeutics. Different proteomic approaches have reported the identification of over 1,300 proteins in saliva. However there are fewer than 100 high abundance proteins, identified by multiple methods including, two-dimensional polyacrylamide gel electrophoresis and HPLC combined with mass spectrometry. Analysis of the genes coding for the salivary proteins demonstrated a non-uniform chromosomal distribution with chromosome 4 having the largest proportion of genes expressed in salivary glands. Several diseases are associated with salivary disorders including Sjögren’s syndrome, Prader-Willi syndrome, dental caries and stress related disorders. Saliva as a diagnostic medium for various biochemical tests has provided a non-invasive and accessibility advantage over other more regularly tested body fluids such as blood and urine. To-date the emerging saliva-based therapeutics include artificial salivas and antimicrobial agents based on histatins and mucins.     

The Potential Anti-Herbivory Role of Microorganisms on Plant Thorns

Thorns, spines and prickles are some of the anti-herbivore defenses that plants have evolved. They were recently found to be commonly aposematic (warning coloration). However, the physical anti-herbivore defense executed by these sharp structures seems to be only the tip of the iceberg. We show that thorns of various plant species commonly harbor an array of aerobic and anaerobic pathogenic bacteria including Clostridium perfringens the causative agent of the life-threatening gas gangrene, Bacillus anthracis, and Pantoea agglomerans. Septic inflammation caused by plant thorn injury can result not only from bacteria. Medical literature indicates that thorns, spines or prickles also introduce pathogenic fungi into animals or humans. Dermatophytes that cause subcutaneous mycoses are unable to penetrate the skin and must be introduced into the subcutaneous tissue by a puncture wound. The common microorganism-thorn combinations seem to have been an important contributor to the fact that so many plant thorns are aposematically colored, as a case of convergent evolution of aposematism in these organisms.Key Words: aposematism, herbivory, pathogen, spine, thorn, bacillus anthracis, clostridium perfringens, sporotrichosis, Mycetoma, subcutaneous mycotic disease     

Atomic force microscopy measurement of leukocyte-endothelial interaction

Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40-100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor-alpha-activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against beta1-integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti-alphaVbeta3. Interestingly, adhesion was also inhibited in a dose-dependent manner (IC50 approximately 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti-beta1 and anti-alphaVbeta3 antibodies. In conclusion, these data demonstrate the role played by beta1-integrins in leukocyte-endothelial adhesion and transmigration and the role played by alphaVbeta3 in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes.     

Segregation of the yeast plasmid: similarities and contrasts with bacterial plasmid partitioning

The high copy yeast plasmid 2 microm circle, like the well-studied low copy bacterial plasmids, utilizes two partitioning proteins and a cis-acting 'centromere'-like sequence for its stable propagation. Functionally, though, the protein and DNA constituents of the two partitioning systems are quite distinct. Key events in the yeast and bacterial segregation pathways are plasmid organization, localization, replication, 'counting' of replicated molecules and their distribution to daughter cells. We suggest that the two systems facilitate these common logistical steps by adapting to the physical, biochemical, and mechanical contexts in which the host chromosomes segregate.     

Photometric assay for measuring the intracellular concentration of branched-chain amino acids in bacteria

The changes in intracellular pool of branched-chain amino acids (BCAA) regulate different physiological processes in bacteria. Up to date, the only available photometric test for measuring BCAA concentration was adapted for blood and plasma samples in diagnostic purposes. We have modified this method for use on bacterial cells, and tested its applicability on several model organisms: Lactococcus lactis, Bacillus subtilis and Escherichia coli.     

How tyrosine phosphorylation affects the UDP-glucose dehydrogenase activity of Bacillus subtilis YwqF

The UDP-glucose dehydrogenase activity of Bacillus subtilis YwqF is regulated by reversible phosphorylation on a tyrosine residue. This reaction, which is catalyzed by the protein-tyrosine kinase YwqD, activates the enzyme, while dephosphorylation of phosphotyrosine-YwqF by the phosphotyrosine-protein phosphatase YwqE reduces its enzyme activity. Our kinetic data indicate that the phosphorylated and unphosphorylated forms of YwqF differ in binding the substrates. The UDP-glucose dehydrogenase reaction catalyzed by YwqF is inhibited by one of its substrates, UDP-glucose, and the extent of this inhibition seems to be reduced upon YwqF phosphorylation. We propose that this effect could at least partly account for the observed activation of YwqF induced by tyrosine phosphorylation. Potential physiological implications of this finding are discussed.