Andragogy in Practice: Applying a Theoretical Framework to Team Science Training in Biomedical Research.

This study is the first to apply the theoretical principles of Malcolm Knowles' theory of andragogy to evaluate data collected from learners who participated in team science training workshops in a biomedical research setting. Briefly, andragogy includes six principles: the learner's self-concept, the role of experience, readiness to learn, orientation to learning, the learner's need to know, and intrinsic motivation. Using an embedded study design, the primary focus was on qualitative data, with quantitative data complementing the qualitative findings. The deductive analysis demonstrated that approximately 85% of the qualitative data could be connected to at least one andragogical principle. Participant responses to positive evaluation questions were largely related to two principles: readiness to learn and problem-based learning orientation. Participant responses to negative questions were largely connected to two different principles: the role of experience and self-direction. Inductive analysis found an additional theme: meeting biological needs. Quantitative survey results supported the qualitative findings. The study findings demonstrate that andragogy can serve as a valuable construct to integrate into the development of effective team science training for biomedical researchers.

Purification of kinase activity from primate parotid glands.

Salivary secretions contain phosphoproteins that contain phosphorylation sites composed of serine residues in acidic environments. The hypothesis of this study is that a protein kinase responsible for phosphorylating these proteins is similar to kinases that phosphorylate proteins in other glandular secretions. Homogenates and subfractions from macaque parotid glands were able to phosphorylate synthetic peptide substrates containing each of the phosphorylation sites in acidic proline-rich proteins, statherin, and histatin 1. Activity was purified from Golgi membranes to greater than 220-fold by extraction with Triton X-100 and affinity chromatography with the use of immobilized ATP. The enzyme preferred substrates containing serine residues in a specific acidic environment, particularly those containing the Ser-Xaa-acidic sequence, preferred ATP over GTP, and was sensitive to high concentrations of heparin. These characteristics are similar to those reported for Golgi casein kinase, which phosphorylates casein in vivo. Based on these observations, the parotid gland kinase may be related to other Golgi-localized serine kinases.

Parotid salivary basic proline-rich proteins inhibit HIV-I infectivity.

OBJECTIVE: The objective of this study was to investigate the molecular nature, spectrum of activity and mechanism(s) of action of those human parotid basic proline-rich proteins that exhibit anti-HIV-I activity. DESIGN: Fractions containing the basic proline-rich proteins were obtained from human parotid saliva of presumed HIV-I non-infected human subjects and characterized with respect to their purity, apparent molecular size and their ability to inhibit the infectivity of T-tropic and M-tropic strains of HIV-I. SUBJECTS, MATERIALS AND METHODS: Stimulated parotid saliva samples were collected from human subjects who denied having any risk factors for HIV-I infection and whose parotid salivas inhibited HIV-I infectivity. Such samples were subjected to affinity, molecular sieve and ion exchange chromatography to isolate individual salivary components. Those fractions demonstrating anti-HIV-I activity were analyzed by SDS-PAGE in order to assess their purity and determine their apparent molecular weights. HIV-I inhibitory activity was determined using HIV-I strains LAI and BaL in a Hela cell-derived multinuclear activation of a galactosidase indicator (MAGI) assay. Amino acid analyses were performed on some fractions. RESULTS: Recombinant gp120-CH-Sepharose chromatography of one subject's parotid saliva revealed specific binding of human parotid basic proline-rich proteins, most prominently one with an apparent molecular weight of 37 kDa. Molecular sieve and cation exchange chromatography yielded a fraction greatly enriched in this protein which amino acid analysis confirmed was proline-rich. A similar fraction from two other subjects also contained basic proline-rich proteins of similar molecular size. These fractions inhibited both T-tropic and M-tropic strains of HIV-I when assayed in the MAGI system. Since SLPI activity is not observable in the MAGI assay, this inhibition was not due to SLPI. The presence of thrombospondin-I (TSP-I) in the active fractions was precluded on the basis of SDS-PAGE examination. CONCLUSIONS: Specific basic proline-rich proteins in human parotid saliva possess significant anti-HIV-I activity independent of that attributable to SLPI or TSP-I. Since the inhibition is detectable with the MAGI assay, its mechanism of action involves virus-host cell interaction prior to the introduction of the tat gene product into the host cell and may be through the binding of the basic proline-rich proteins to the HIV-I gp120 coat of the virus.

Electron-microscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles in vitro.

Proline-rich proteins (PRPs), histatins, and statherin are salivary proteins that exhibit high affinities for hydroxyapatite surfaces. In vitro experiments with parotid submandibular/sublingual or whole saliva have shown these proteins to adsorb selectively to tooth surfaces. This investigation focuses on the histo-morphological identification of PRPs, histatins, and statherin in acquired enamel pellicles. Synthetic hydroxyapatite or bovine enamel were exposed to glandular secretions, and whole saliva and pellicle precursor proteins were identified immunohistologically by electron microscopy. Results obtained by back-scattered scanning electron microscopy showed these proteins to be present in pellicles. Pellicles displayed a distinct structure consisting of a sponge-like meshwork of microglobules. Interconnections between structural elements were identified in submandibular/sublingual and whole saliva pellicles only. Transmission electron microscopy of pellicles formed on bovine enamel surfaces revealed a tendency for preferential localization of precursor proteins within the protein film. Since the data showed the presence of pellicle precursors in pellicles derived both from glandular secretions and from whole saliva, it is likely that PRPs, histatins, and statherin are integral components of acquired enamel pellicles in vivo.

New in vitro model for the acquired enamel pellicle: pellicles formed from whole saliva show inter-subject consistency in protein composition and proteolytic fragmentation patterns.

The present investigation was undertaken to investigate the variability of proteins in whole saliva which adsorb to hydroxyapatite and are thus likely to be precursors of the acquired enamel pellicle. Whole-saliva proteins from 18 subjects were absorbed to hydroxyapatite, and the gel filtration patterns of released proteins revealed four major peaks and three minor peaks eluting between the major peaks. Amino acid analysis indicated that minor peaks contained fragments of proteins in major peaks, and this was confirmed by sequence analysis. Major peaks comprised 95% and minor peaks comprised 5% of protein absorbed to hydroxyapatite, suggesting a limited proteolytic capacity of whole saliva. HPLC elution patterns of components in minor peaks suggested that proteolysis is not totally random but is an orderly and consistent process. These studies suggest that whole saliva may be suitable as a model system for the investigation of post-secretory modifications of salivary proteins important for the formation of the acquired enamel pellicle.

Pellicle precursor protein crosslinking characterization of an adduct between acidic proline-rich protein (PRP-1) and statherin generated by transglutaminase.

Recent work with oral transglutaminase indicated that this enzyme, derived from oral epithelial cells, crosslinked pellicle precursor proteins which may be important in the formation of the acquired enamel pellicle. The purpose of this study was to investigate whether purified acidic PRP-1 can form crosslinks with statherin, and whether such a crosslink is derived from a transglutaminase-catalyzed reaction between glutaminyl and lysyl side-chains, leading to a covalent bond formation. Enzymatic reaction products were analyzed by SDS-PAGE and reverse-phase HPLC. The SDS electrophoretogram revealed a protein band with an apparent molecular weight of 32 kDa, which is consistent with the combined apparent molecular weight of acidic PRP-1 (24 kDa) and statherin (8 kDa). A reaction product isolated by HPLC was characterized by amino acid analysis, which showed a stoichiometry consistent with being an adduct composed of one molecule of acidic PRP-1 and one molecule of statherin. In negative control experiments, it could be shown that this adduct was not detected when the lysines of both substrates were modified by reductive methylation prior to the enzymatic reaction. In addition, amino acid analysis and mass spectrometry confirmed the presence of a gamma-glutamyl-epsilon-lysine dipeptide after enzymatic hydrolysis and the absence of this dipeptide after acid hydrolysis. Analysis of the data obtained indicates that oral transglutaminase is capable of crosslinking acidic PRP-1 and statherin in vitro. In addition, this finding exemplifies the potential of post-secretory processing of salivary proteins, which may represent an additional mechanism to generate new protein species.

Pellicle precursor proteins: acidic proline-rich proteins, statherin, and histatins, and their crosslinking reaction by oral transglutaminase.

Previous studies have demonstrated that whole saliva and pellicle formed in vitro from oral fluid contain covalently crosslinked salivary proteins. The purpose of this study was to determine which salivary proteins can act as substrates for transglutaminase, an enzyme responsible for the covalent crosslink reaction between a glutamine residue and a lysine residue. Transglutaminase was prepared from the pellet fraction of human whole saliva. Dansyl cadaverine (N-dansyl-1,5-diaminopentane) was used to study the reactivity of glutamine residues in acidic large and small proline-rich proteins, statherin, and the major histatins, whereas a glutamine-containing dansylated peptide was used to study the reactivity of lysine residues in these proteins. Crosslink formation was measured fluorometrically after the addition of fluorescent probe to the salivary protein substrate and transglutaminase. The covalent attachment of the fluorescent probe to salivary proteins was confirmed by SDS-PAGE. It was found that almost all of the lysines present in the acidic PRPs and statherin, and some of the lysines present in histatins, could participate in the crosslink reaction. Glutamine reactivity was also observed, but a maximum of only 14% of glutamine residues present in acidic PRPs and statherin participated in the crosslink formation. These results demonstrate that primary pellicle precursor proteins, acidic proline-rich proteins, statherin, and the major histatins are capable of undergoing crosslink reactions catalyzed by oral transglutaminase. This may enable other proteins in the oral cavity to be incorporated into the acquired enamel pellicle.

Salivary secretions: narcolepsy and central nervous system stimulants.

In an ongoing attempt to develop a model to study the influence of various diseases and drugs on saliva, we studied persons with narcolepsy treated with central nervous system stimulants. The aim was to study the secretion of salivary proteins in narcolepsy in the presence and absence of central nervous system stimulants. For this purpose, two proteins synthesized in acinar cells, acidic proline-rich proteins (PRPs) and statherin, were selected. Persons with narcolepsy treated with central nervous system stimulants only were included, n = 12, ages 14 to 68, seven females. Citric-acid-stimulated parotid and submandibular/sublingual saliva samples were collected from these persons during medication, after a drug-free period of one week, at least two weeks after the drug had been re-introduced, and from a matched healthy control group. PRP and statherin concentrations were determined by means of reversed-phase and anion exchange chromatography, respectively. Both concentration and output of statherins and PRPs were increased in persons with narcolepsy receiving central nervous system stimulants compared with healthy control individuals. When the drug was withdrawn, salivary flow rates were not influenced. In contrast, withdrawal of the drug led to a significantly decreased secretion of PRPs and statherin. The reduced protein secretion may reflect decreased adrenergic activation in narcolepsy, to be reversed by treatment with central nervous system stimulants. It can be concluded that measurements of both salivary fluid and salivary proteins may be necessary for an overall evaluation of the effects of a given drug or disease on salivary secretion.

In vitro elution of leachable components from dental sealants.

Recent concerns have been raised about the possibility that estrogenic chemicals, in particular bisphenol-A, or BPA, might be leached out of dental sealants. This study aimed to identify and quantify BPA and other components released from seven light-cured fissure sealants in vitro. None of the tested sealants was shown to have released BPA; however, the investigators identified other eluted components that should be investigated for their biological effects.

Temporal and compositional characteristics of salivary protein adsorption to hydroxyapatite.

Salivary proteins bind to enamel surfaces and hydroxyapatite in a highly selective manner. Numerous studies have identified these proteins as primarily proline-rich proteins, cystatins, statherin, and histatins. Previously, the hydroxyapatite-binding potential of these proteins had been characterized in systems consisting of singly purified protein and adsorbent. The purpose of this study was to investigate the adsorption of each protein in the presence of complete salivary secretion. Proteins, shown to adsorb to hydroxyapatite, were purified, biotinylated, and added back to the remaining proteins to form a series of reconstituted secretions. The adsorption of each biotinylated protein in the reconstituted secretion to hydroxyapatite was then measured as a function of time. Results indicated that three different adsorption patterns occur. A simple hyperbolic pattern is characteristic of amylase, glycosylated proline-rich protein (PRG), and cystatin. A faster adsorption process is observed for PRP-3, PRP-4, PIF-f, and statherin. A more complex pattern, exhibiting a rapid phase followed by a slower phase, is characteristic of PRP-1, PRP-2, PIF-s, and histatins. These results suggest that there are different adsorption processes involved in the binding of salivary proteins to hydroxyapatite. Two possible mechanisms are direct adsorption of protein to hydroxyapatite and indirect adsorption of protein by interacting with other proteins already bound to hydroxyapatite.

Physiological regulation of the secretion of histatins and statherins in human parotid saliva.

The small salivary phosphoproteins, histatins and statherins, have important functions in the oral cavity in terms of antimicrobial actions and regulation of calcium phosphate homeostasis. Neither the effects of various physiological stimuli on their secretion nor the nature of the efferent receptor involved in the stimulus-secretion coupling has been determined previously. These aspects are important for improved understanding of the secretory control of salivary proteins and may have implications regarding the effects of specific medications on salivary constituents and oral health. The effects of graded mechanical (chewing on short and long silicone tubings) and gustatory stimulation (0.5, 1.5, and 5.0% citric acid) on the secretion of histatins and statherins were studied in the presence and absence of adrenolytic agents (n = 10). In this model, secretory rates of both proteins increased with increases in flow rate, with 5.0% citric acid representing a particularly potent stimulus. Histatin and statherin secretory rates were significantly reduced by the beta 1-adrenolytic agent (histatins to 58 to 72% and statherins to 11 to 29% of that in corresponding control experiments), but not by the alpha 1-adrenolytic agent. Since the beta 1-adrenergic receptors played an important role in the stimulus-secretion coupling of these proteins, protective salivary functions in the oral cavity may be compromised during beta 1-adrenolytic treatment.

Antisense RNA to the first N-glycosylation gene, ALG7, inhibits protein N-glycosylation and secretion by Xenopus oocytes.

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

Structural features of salivary function.

Saliva plays an important role in the maintenance of oral health by exhibiting multiple host defense functions. These include homeostatic processes, lubrication, antimicrobial activity, and the control of demineralization/remineralization of teeth. Biochemical studies of saliva and salivary secretions established that specific salivary proteins are responsible for these defense functions. Because some of these salivary proteins have been characterized extensively, including their primary structures, it has become feasible to explore their structure/function relationships. Acidic proline-rich proteins (PRPs), for example, exhibit high affinity to hydroxyapatite, inhibit crystal growth of calcium phosphate salts from solutions supersaturated with respect to hydroxyapatite, bind calcium ions, and interact with several oral bacteria on adsorption to hydroxyapatite. Statherins, histatins, and cystatins also exhibit affinities to mineral surfaces, inhibit calcium phosphate precipitation, and play a role in maintaining the integrity of teeth. Furthermore, histatins exhibit both antibacterial and antifungal activities. Approaches to identifying the functional domains of these salivary proteins include functional assays of enzymatically digested proteins and peptides, synthetic peptides and peptide analogues, and chemically modified proteins as well as biophysical studies of native proteins or peptides. Such studies have demonstrated that the fungicidal activities of histatins reside in the middle portion of the polypeptide chain, whereas the hydroxyapatite binding domains of PRPs and statherin reside in the phosphorylated amino-terminal regions. Identification of functional domains is vital in understanding the mechanisms of action and this information can be exploited in the development of therapeutic agents.

Adsorption of human salivary proteins to hydroxyapatite: a comparison between whole saliva and glandular salivary secretions.

The protein compositions of in vitro pellicles formed from whole saliva and parotid and submandibular secretions were determined by use of synthetic hydroxyapatite as a model for dental enamel. The adsorbed and unadsorbed protein fractions were analyzed by amino acid analysis and both anionic and cationic discontinuous polyacrylamide gel electrophoresis. For further characterization of the in vitro pellicle, the adsorbed fractions were subjected to gel filtration on Sephadex G-100 and reversed-phase chromatography on C18 columns. Amylase, acidic and glycosylated proline-rich proteins, statherins, and histatins were identified in the parotid-derived pellicle. Detailed analysis of the statherin-containing fractions resulted in the observation of several statherin-like proteins. The use of cationic gel electrophoresis allowed for the identification of histatin 3 and histatin 5, which have not been previously detected in pellicle formed in vitro. The protein composition of submandibular-derived pellicle was similar to that of parotid-derived pellicle except for the presence of cystatins and the absence of glycosylated proline-rich proteins. In contrast, in vitro pellicle derived from whole saliva exhibited a vastly different composition, consisting primarily of amylase, acidic proline-rich proteins, cystatins, and proteolytically-derived peptides. The results indicate that acidic phosphoproteins as well as neutral and basic histatins from pure secretions selectively adsorb to hydroxyapatite, whereas in whole saliva some of these proteins are proteolytically degraded, dramatically changing its adsorption pattern.

Salivary cystatin SA-III, a potential precursor of the acquired enamel pellicle, is phosphorylated at both its amino- and carboxyl-terminal regions.

Cystatin SA-III was purified from human submandibular/sublingual glandular secretions by adsorption to hydroxyapatite, gel filtration chromatography, and reversed-phase HPLC. The amino acid sequence of its amino-terminus was deduced by sequential Edman degradation and found to be identical to the first 10 residues of cystatin HSP-12. The purified protein was digested with endoproteinase Asp-N and the digestion products were subjected to fast atom bombardment mass spectroscopy. m/z values corresponding to 12 peptides were aligned to the sequence of cystatin S preceded by the eight-residue amino-terminal peptide detected in HSP-12. This process resulted in the assignment of peptides corresponding with 118 out of the 121 amino acid residues predicted from the nucleotide sequence for cystatin SA-III. In order to align several peptides, it was necessary to substitute four residues of phosphoserine for four residues of serine. Fast atom bombardment mass spectrometry and additional Edman degradation procedures localized the phosphate moieties to Ser-3, Ser-99, Ser-112, and Ser-116. This is the first report of the structure of cystatin SA-III deduced by amino acid sequencing techniques and indicates the sites of phosphoserine within the molecule. Based on these assignments, cystatin SA-III is unique among salivary proteins in that it possesses phosphate groups at its amino-terminus as well as its carboxyl-terminus.

Multiple forms of statherin in human salivary secretions.

Sequential chromatography of hydroxyapatite-adsorbed salivary proteins from submandibular/sublingual secretions on Sephadex G-50 and reversed-phase HPLC resulted in the purification of statherin and several statherin variants. Amino acid analysis, Edman degradation and carboxypeptidase digestion of the obtained protein fractions led to the determination of the complete primary structures of statherin SV1, statherin SV2, and statherin SV3. SV1 is identical to statherin but lacks the carboxyl-terminal phenylalanine residue. SV2, lacking residues 6-15, is otherwise identical to statherin. SV3 is identical to SV2 but lacks the carboxyl-terminal phenylalanine. These results provide the first evidence for multiple forms of statherin which are probably derived both by post-translational modification and alternative splicing of the statherin gene.

Excitation energy transfer study of the spatial relationship between the carbonyl and metal cofactors in pig plasma amine oxidase.

9-Hydrazinoacridine irreversibly labeled pig plasma amine oxidase by covalent attachment to the active carbonyl cofactor. The visible absorption spectrum of the modified protein displays new absorption bands at 495 and 525 nm. Its emission spectrum exhibited maxima at 415 and 440 nm. In addition, both absorption and emission spectra were insensitive to pH changes between 6 and 10. Phase modulation fluorometry was used to determine fluorescence lifetimes of Zn2+- and Co2+-substituted acridinyl plasma amine oxidase. Energy transfer efficiency was 22%; the distance separating the Co2+ ion (in the copper binding site) and the acridine moiety (the amine substrate binding site) ranges between 11.7 and 14.7 A. This work defines the proximity of the metal and substrate (and hence the carbonyl cofactor) and precludes any direct interaction between Cu2+ and pyrroloquinoline quinone or between Cu2+ and the substrate.

Human serum alpha 2HS-glycoprotein modulates in vitro bone resorption.

We previously isolated a family of bone-resorbing proteins from human cancer ascites fluid and established that the three purified bone-resorbing proteins were chemically and immunochemically related to each other and to alpha-2HS glycoprotein (alpha 2HS). After this finding we purified the normal human serum counterpart of these ascites proteins and studied its effects on bone resorption. The bone-resorbing properties of normal human serum alpha 2HS were examined in vitro over a wide dose range. This normal human serum glycoprotein had a biphasic effect on 45Ca2+ release from bone. More specifically, this protein stimulated bone resorption at the lower concentrations tested, with a maximum effect [treated over control ratio of 2.5 +/- 0.30 (+/- SE); P less than 0.01] at 40 micrograms/mL. In contrast, at doses above 40 micrograms/mL, a sharp decline in calcium mobilization occurred, with a return to baseline occurring above 80 micrograms/mL. These results suggest that serum alpha 2HS may participate in the regulation of bone metabolism in vivo.

Three forms of BRP-2 (bone resorptive proteins) from human cancer ascites fluid and their relationship to human serum alpha-2 HS-glycoprotein.

Two new forms of BRP-2, a previously described bone resorptive protein, were purified from ascites fluids obtained from patients with hypercalcemia and metastatic bone cancer. The apparent molecular weights of BRP-2 and of these two proteins were 52,000, 48,000, and 46,000, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The three proteins have essentially the same amino acid compositions but differ with respect to their carbohydrate moieties. The amino-terminal amino acid sequences of the three glycoproteins were identical to each other as well as to human serum alpha 2HS-(human serum) glycoprotein. The relationship of the three forms of BRP-2 to alpha 2HS was also established immunochemically. The ascites proteins, as well as alpha 2HS, on a molar basis, were approximately one-tenth as potent as bovine parathyroid hormone fragment (1-34) in their abilities to stimulate calcium release from bone in vitro. This study describes for the first time a possible function for human serum alpha 2HS.

Site-specific photo-cross-linking studies on interactions between troponin and tropomyosin and between subunits of troponin.

We have used the sulfhydryl-specific heterobifunctional photo-cross-linker 4-maleimidobenzophenone (BP-Mal) to study the interactions of rabbit skeletal tropomyosin with troponin and of the troponin subunits with each other. We found that alpha,alpha-tropomyosin specifically labeled at Cys-190 with BP-Mal photo-cross-links with all three subunits of troponin with decreasing cross-linking yields in the order of troponin T, troponin I, and troponin C. There was no apparent Ca2+ dependence in the cross-linking yields. In separate experiments, we found that troponin C labeled specifically at Cys-98 with BP-Mal photo-cross-links to both troponin I and troponin T in the two binary complexes, as well as in the ternary complex. Again, no Ca2+-dependent changes in the cross-linking yields were detectable. These results are in general agreement with the picture that troponin I and troponin T are in close contact with troponin C near its Cys-98 and that all three troponin subunits are in the proximity of Cys-190 of tropomyosin.