Physiological concentration of human salivary histatins in glandular secretions and whole saliva

Includes bibliography: (leaves 114-124).Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1996 (Oral Biology).Histatins are a group of histidine-rich antimicrobial proteins present in human salivary secretions. Previous studies have shown that histatins play an important role in the maintenance of enamel integrity and defense against oral bacteria and fungal pathogens in the oral cavity. Because histatins differ in their ability to inhibit blastopore viability and germ tube formation, it is important to be able to quantitatively determine individual histatin concentrations. Such determinations would be essential for understanding and comparing the antibacterial activities of the saliva in healthy and diseased persons, and in finding a relation between individual histatin concentration and function. The present study is focused on determination of the physiological concentration of the major histatins 1, 3, and 5 in glandular secretions and whole saliva in order to evaluate the relationship of individual histatin concentrations to their functional capacity in the oral cavity. Human parotid and submandibular / sublingual secretions were collected from 19 healthy donors in the presence and absence of gustatocy stimulation. Whole saliva was collected from the same group of donors with masticatocy stimulation. A cationic polyaccylamide gel electrophoresis system (cationic PAGE) was used in combination with scanning densitometcy to measure the [TRUNCATED]

The bactericidal effects of human salivary histatin 5 on Porphyromonas gingivalis

Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1994 (Oral Biology).Includes bibliographical references (leaves 57-84).Previous studies with oral bacterial and fungal pathogens have demonstrated that histatin 5 is the most potent histatin, a group of histidine-rich salivary proteins present in parotid secretions with respect to antibacterial and antifungal effects. The present study was aimed at investigating the ef feet of human parotid salivary histatin? on the viability of the periodontal pathogen Porphyromonas gingivalis (P.g.). Experimentally, the viability of P.g. was studied after exposure to incremental doses of histatin 5 at different pH values (pH 5.0, 6.0, 7.0 and 8.0). Histatin 5 from human parotid secretion was isolated by Bio-Gel P-2 chromatography and reverse-phase high-performance liquid chromatography. The purity of histatin 5 was determined by polyacrylamide gel electrophoresis and amino acid analysis. Following incubation, bacteria were washed by successive centrifugation, plated onto enriched Trypticase soy agar, and incubated anaerobically at 37 °C. After 48 hr, colony forming units were counted. Linear regression analysis was used to evaluate doseresponse. The most perfect fit for a linear regression was found for pH 6.0 and 7.0 (p<0.02) with corresponding LD50 values of 0.19 mM and 0.51 mM, respectively. The data indicate that histatin 5 is bactericidal on P.gingivalis and this effect is modulated by the pH values of the bacterial environment in contact with histatin 5. The relative bactericidal potency was higher at pH 5.0 and decreased as pH increased

Protein Inhibitors of Calcium Salt Crystal Growth in Saliva, Bile and Pancreatic Juice

The control of the formation of crystals in biological fluids is one of the most exciting field of research involving both organic and biochemical areas. Many organisms have evolved mechanisms which minimize or avoid the effects of nucleation and crystal growth formation. One of the most important mechanism is the interaction of specific proteins, called inhibitors, with crystals which alters their habits and leads to their elimination. This article, focused on saliva, pancreatic juice and bile, reviews our present knowledge on the structure-function relationships existing between these proteins and their ability to inhibit the growth of different calcium salt crystals

Top-down platform for deciphering the human salivary proteome

Proteomic platforms can be classified in bottom-up strategies, which analyze the sample after proteolytic digestion, and top-down strategies, which analyze the intact naturally occurring proteome. Bottom-up platforms are high-throughput because they can investigate a large number of proteins, regardless of their dimension. Nonetheless, information on post-translational modifications (PTMs) can be lost, especially those regarding naturally occurring cleavages and alternative splicing. Top-down platforms cannot cover vast proteomes, however, they can disclose subtle structural variations occurring during protein maturation and allow label-free relative quantifications in an unlimited number of samples. A repertoire of 256 masses belonging to naturally occurring proteins and peptides consistently detected by RP-HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva is presented in this study. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. The present review reports average and mono-isotopic masses of the peptides and proteins detected, RP-HPLC elution times, PTMs, origin and quali-quantitative variations observed in several physiological and pathological conditions. The information reported can be a reference for users of top-down RP-HPLC-ESI-MS proteomic platforms applied to the study of the human salivary proteome as well as of other human bodily fluids

The binding of salivary proteins and its effect on the demineralization of hydroxyapatite

Thesis (M.A.)--Boston UniversityHistatins, statherin and proline-rich proteins (PRPs) are important members of the human saliva proteome. They are components of the acquired enamel pellicle (AEP) which provides protection to the teeth and perform other vital functions in the oral cavity such as preventing spontaneous precipitation of calcium phosphate salts (statherin) and exhibiting fungicidal activity (histatins). The aims of the study were to (1) isolate histatins, statherin and acidic PRPs from parotid secretion, (2) develop a novel functional assay for the measurement of hydroxyapatite demineralization by modifying pre-existing methods and (3) test the binding of mixed PS proteins and purified salivary proteins (histatin 1, statherin and PRP1) to HA and the subsequent effect on demineralization. The steps in the histatin, statherin and PRP purification process include zinc precipitation of parotid saliva, ion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). Protein purity was assessed by polyacrylamide gel electrophoresis (PAGE). The functional properties of the proteins were examined in two ways: Binding of mixed and purified proteins to ceramic hydroxyapatite (HA) powder was monitored by PAGE and HPLC, and demineralization of HA exposed to acidic conditions after incubation with mixed and purified proteins was determined in spectrophotometric assays for free calcium and phosphate. Histatins and PRPs were successfully isolated to high levels of purity from human parotid saliva (HPS). Salivary protein binding experiments showed that the order of the three proteins studied in terms of their apparent affinity for HA is: statherin > histatin 1 > PRP1. The linearized Langmuir adsorption plots for histatin 1 and statherin fit well, showing R2 values of 0.9880 and 0.9975, respectively. However, the adsorption isotherm for PRP1 did not exhibit linear characteristics (R2 value of 0.61). Functional assays performed to measure the release of calcium and phosphate ions showed variable levels of protection afforded to HA by preincubation with the three proteins. On a molar basis, histatin 1 provided the most protection against demineralization. The in vitro data generated add to a more complete understanding of the functional characteristics of histatins, statherin and PRP1 and thereby provide insights into their potential capacity to protect enamel in the oral environment

Characterization of promoter regions of parotid gland genes.

BACKGROUND: Salivary secretion aids in digestion, host defense, and lubrication. Parotid salivary gland defects from Sjogren’s Syndrome affect more than one million Americans and cause dry mouth leading to oral disease. The Amylase 1 gene and the PSP gene are markers of parotid gland differentiation and indicate normal parotid gland function. Transcriptional activators of genes involved in parotid differentiation have not been characterized. HYPOTHESIS: Characterizing transcriptional activating sites in gene promoter regions will confirm upstream activating factors in bioinformatic network pathways. METHODS: Two promoter regions of mouse amylase 1 were identified using the ECR Browser, PCR amplified and ligated into a pGL4.10 luciferase vector. Transcriptional repressor binding sites were identified and removed from the 1 kbp region, followed by PCR amplification to produce fragments that were ligated into the minimal promoter pGL4.23 luciferase vector. These promoter clones were then transfected into three cell lines and tested for promoter activity. Bioinformatic analysis of microarray data determined correlations between increased transcription factor expression and markers of parotid differentiation. A hypothetical transcription factor network was created. Two interactions from the network predicted to activate the NUPR1 gene were Cited2/p300 and IRF2/PCAF. Mist1 was suggested to activate the PSP gene based on the hypothetical network. The promoter regions of NUPR1 and PSP were transfected into a parotid acinar cell line, and promoter activity was determined using dual luciferase assays with an internal Renilla control. RESULTS: The four amylase promoter clones, R1, R2-R1, 900bp, and 700bp were repressed as compared to a promoterless negative control, pGL4.10. The NUPR1 promoter was not activated by the co-transfected combination of Cited2/p300 or IRF2/PCAF. The PSP promoter alone was not activated by Mist1 transcription factor. However, PSP was activated when Mist1 was co-transfected with Tcf3 transcription factor and when intron regions containing E-box binding sites were added to the PSP promoter clone. CONCLUSION: Amylase 1 was not activated in the regions tested; therefore the active regions of the amylase 1 gene must be outside the 3 kbp region tested. NUPR1 is not activated by the predicted interactions of Cited2/p300 or IRF2/PCAF, so the results do not support the hypothetical transcription factor network. The PSP proximal promoter is not activated by Mist1, but a PSP promoter + intron construct is activated when the transcription factor Tcf3 + Mist1 are co-transfected

Identification of the Salivary Proteome in Children Throughout the Course of Dental Eruption

The salivary proteome is recognized as a valuable source of potential oral and systemic disease biomarkers. Major efforts in salivary research have been dedicated to identify and characterize salivary proteins present in saliva using both classical biochemical methods and proteomics approaches in adults. Despite considerable research on the salivary proteome, little attention has been given to the changes in the salivary proteome occurring in children, specifically from 0-3 years of age. Through the use of anionic PAGE, SDS PAGE, HPLC and MS/MS, salivary protein profiles in children before, during and after dental eruption were compared with edentulous adult controls. We identified substantive qualitative and quantitative differences in the salivary proteome between children and adults, suggesting a greater emphasis is warranted in the study of the changes in the salivary proteome as a function of age and dental status

Acquired Bracket Pellicle Modulation Via Exposure To Histatin 3

Objectives: To investigate the effect of histatin 3 on the protein pellicle formation on the orthodontic bracket surface. Methods: Our study consisted of 4 sample groups. A sample of human parotid saliva without histatin functioned as a control group. Other groups were immersed with or without histatin 3 and human parotid saliva. Each group was incubated for 2 hours in their respective substrate at 37°C. The acquired pellicle from each group was harvested, then analyzed with SDS-PAGE and LC-ESI-MS/MS. Results: Thirty-nine proteins were identified in the control group, 18 were identified in group 2, and 21 were identified in group 3. Thirteen proteins were common to all groups. Groups immersed in histatin 3 and human parotid saliva showed an increase in the percentage of proteins exhibiting antimicrobial activities and immune response. Conclusions: There were functional differences in the protein pellicle formed on the orthodontic bracket, suggesting that exposure to histatin 3 may alter pellicle formation. However, structural differences were limited due to redundant characteristics of salivary proteins