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.     

Effects of temperature on the binding of acid and basic dyes,including reference to metachromasy

Summary With a number of anionic dyes, a positive temperature effect occurs in the staining of tissues, whereas with a number of cationic dyes, a negative temperature effect occurs. The positive effect involves increased dye binding at 45⁰ as compared to 5⁰; the negative effect involves decreased dye binding at the higher temperature. To obtain these effects, dye concentration must be low and staining must be continued to equilibrium, i. e., for about 24 hours. These facts suggest that the temperature effects may depend in part on the degree of ionization of tissue components and also on competition between tissue components and dye for chromotrope.Deamination of sections depresses acidophilia and enhances basophilia but fails to obliterate the temperature effects.With metachromatic basic dyes, despite reduction in staining of highly acidic compounds at high temperature, the color remains metachromatic. This result differs from that obtained in the test tube and is probably explained by the fact that the chromotropes are relatively fixed in position in tissue sections.

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Zusammenfassung Bei Gewebsfärbungen lassen einige anionische Farbstoffe eine positive Temperaturwirkung erkennen, d. h. eine Zunahme der Farbstoffbindung beim Steigen der Temperatur von 5⁰ auf 45⁰. Bei kationischen Farbstoffen liegt dagegen meist eine negative Temperaturwirkung vor, d. h. eine Verminderung der Farbstoffbindung bei höherer Temperatur. Dieser Effekt kann nur bei niedriger Farbstoffkonzentration und längerer Färbedauer (etwa 24 Std) erzielt werden. Die Temperaturwirkung hängt wohl z. T. vom Grad der Ionisation der Gewebsbestandteile ab wie vom Wettbewerb zwischen Gewebsbestandteil und Farbstoff für Chromotrope.Desaminierung vermindert die Acidophilie und steigert die Basophilie entsprechender Gewebebezirke. Die Temperaturwirkung bleibt erhalten.Trotz Minderung der Anfärbbarkeit von stark sauren Verbindungen bei hoher Temperatur bleibt die Metachromasie mit metachromatisch wirkenden basischen Farbstoffen im histologischen Schnitt erhalten. Im Reagensglasversuch liegen andere Ergebnisse vor. Dieser Unterschied erklärt sich wahrscheinlich aus der ziemlich festen Bindung zwischen Chromotrop und Gewebe.

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With 8 Figures in the Text, of which 1 in colour

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This research was supported in part by grants from the United States Public Health Service (H-2190, C2) and from the Dickinson Research Memorial, Planned Parenthood Federation of America, 501 Madison Avenue, New York 22, N. Y.     

Tetracycline-inducible gene regulation in mycobacteria

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml⁻¹ of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.     

A radiation-induced acute apoptosis involving TP53 and BAX precedes the delayed apoptosis and neoplastic transformation of CGL1 human hybrid cells

Exposing CGL1 (HeLa x fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of approximately 50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-X(L) is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.     

Dynamic Histone Acetylation of Late Embryonic Genes during Seed Germination

Histone acetylation is involved in the regulation of gene expression in plants and eukaryotes. Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from histones, which is associated with the repression of gene expression. To study the role of histone acetylation in the regulation of gene expression during seed germination, trichostatin A (TSA), a specific inhibitor of histone deacetylase, was used to treat imbibing Arabidopsis thaliana seeds. GeneChip arrays were used to show that TSA induces up-regulation of 45 genes and down-regulation of 27 genes during seed germination. Eight TSA-up-regulated genes were selected for further analysis – RAB18, RD29B, ATEM1, HSP70 and four late embryogenesis abundant protein genes (LEA). A gene expression time course shows that these eight genes are expressed at high levels in the dry seed and repressed upon seed imbibition at an exponential rate. In the presence of TSA, the onset of repression of the eight genes is not affected but the final level of repressed expression is elevated. Chromatin immunoprecipitation and HDAC assays show that there is a transient histone deacetylation event during seed germination at 1 day after imbibition, which serves as a key developmental signal that affects the repression of the eight genes. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Prime-boost immunisation strategies for tuberculosis

Vaccines against intracellular pathogens such as Mycobacterium tuberculosis need to induce strong cellular immune responses. Heterologous prime-boost immunisation strategies induce higher levels of both CD4+ and CD8+ T cells than homologous boosting with the same vector. Recombinant pox-viruses are particularly good at boosting previously primed T cell responses. Using BCG as the priming immunisation in such a heterologous prime-boost strategy is a practical solution, which allows the beneficial effects of BCG in children to be maintained.     

A structure-based method for identifying DNA-binding proteins and their sites of DNA-interaction

A classification model of a DNA-binding protein chain was created based on identification of alpha helices within the chain likely to bind to DNA. Using the model, all chains in the Protein Data Bank were classified. For many of the chains classified with high confidence, previous documentation for DNA-binding was found, yet no sequence homology to the structures used to train the model was detected. The result indicates that the chain model can be used to supplement sequence based methods for annotating the function of DNA-binding. Four new candidates for DNA-binding were found, including two structures solved through structural genomics efforts. For each of the candidate structures, possible sites of DNA-binding are indicated by listing the residue ranges of alpha helices likely to interact with DNA.     

The genome and the nucleus: a marriage made by evolution

Genomes are housed within cell nuclei as individual chromosome territories. Nuclei contain several architectural structures that interact and influence the genome. In this review, we discuss how the genome may be organised within its nuclear environment with the position of chromosomes inside nuclei being either influenced by gene density or by chromosomes size. We compare interphase genome organisation in diverse species and reveal similarities and differences between evolutionary divergent organisms. Genome organisation is also discussed with relevance to regulation of gene expression, development and differentiation and asks whether large movements of whole chromosomes are really observed during differentiation. Literature and data describing alterations to genome organisation in disease are also discussed. Further, the nuclear structures that are involved in genome function are described, with reference to what happens to the genome when these structures contain protein from mutant genes as in the laminopathies. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK