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Daily Saliva and Vaginal Mucins Co-vary in Protein-to-Carbohydrate Ratio
Abstract
The currently reported study employed the curiously named (but simple) quantitative, nondestructive technique of Multiple Attenuated Internal Reflection InfraRed (MAIR-IR) spectroscopy to assess daily variations of both saliva and vaginal secretions from a panel of consenting volunteers. The project goal was to determine whether the protein-to-carbohydrate (starch) ratio, determined by MAIR-IR analysis of salivary and vaginal secretions is an objective measure of the co-variation of mucinous secretions with daily menstrual cycle. Overall, the spectral data were found to provide valid signatures of vaginal mucus chemical changes that also correlated with cyclic periods of presumed fertility, such periods exhibiting repeat times of 14-16 days and 27-29 days in the thoroughly analyzed records. At mid-cycle, associated with ovulatory events, the protein-to-carbohydrate ratio of vaginal mucin maximizes. Higher carbohydrate ratios are associated with stiffer cervical mucus plug formation, which usually prevents infection, spontaneous abortion, and pre-term birth, but may also be responsible for infertility. Mucin relaxation compounds, such as the FDA-approved oral rinse, delmopinol, may induce fertility in such cases.
The pericellular matrix is a robust hyaluronan-rich polymer brush-like structure that controls access to the cell surface, and plays an active role in cell adhesion, migration and proliferation. We report the observation of single bottlebrush proteoglycan dynamics in the pericellular matrix of living chondrocytes. Our investigations show that the pericellular matrix undergoes gross extension upon the addition of exogenous aggrecan and that this extension is significantly in excess of that observed in traditional particle exclusion assays. The mean square displacement of single proteoglycans increases with distance to cell surface indicating the openings in the microstructure that are on the order of ~150nm near the cell surface and ~400nm near the PCM edge. In addition, the mobility of tethered aggrecan drops significantly when the cell coat is enriched with bottlebrush proteoglycans. Single molecule imaging in this thick polysaccaride matrix on living cells has significant promise in the drive to elucidate the role of the pericellular coat in human health.
Ascending infection from the colonized vagina to the normally sterile intrauterine cavity is a well-documented cause of preterm birth. The primary physical barrier to microbial ascension is the cervical canal, which is filled with a dense and protective mucus plug. Despite its central role in separating the vaginal from the intrauterine tract, the barrier properties of cervical mucus have not been studied in preterm birth.
To study the protective function of the cervical mucus in preterm birth we performed a pilot case-control study to measure the viscoelasticity and permeability properties of mucus obtained from pregnant women at high-risk and low-risk for preterm birth. Using extensional and shear rheology we found that cervical mucus from women at high-risk for preterm birth was more extensible and forms significantly weaker gels compared to cervical mucus from women at low-risk of preterm birth. Moreover, permeability measurements using fluorescent microbeads show that high-risk mucus was more permeable compared with low-risk mucus.
Our findings suggest that critical biophysical barrier properties of cervical mucus in women at high-risk for preterm birth are compromised compared to women with healthy pregnancy. We hypothesize that impaired barrier properties of cervical mucus could contribute to increased rates of intrauterine infection seen in women with preterm birth. We furthermore suggest that a robust association of spinnbarkeit and preterm birth could be an effectively exploited biomarker for preterm birth prediction.
We report for the first time a masticatory-lubrication assay system to assess the lubricating properties of salivary constituents. The lubricating ability of the proline-rich glycoprotein (PRG) of parotid saliva was enhanced by human serum albumin. The interactive effect of albumin was abolished by chemically deglycosylating the glycoprotein. Fluorescence spectroscopy with a hydrophobic probe verified the existence of a PRG-albumin complex and demonstrated that deglycosylation of the PRG altered the nature of its interaction with albumin.
At least eight mucin apoproteins are expressed by the tracheobronchial epithelium, but it is not known which, if any, of these are major constituents of the respiratory secretions responsible for the formation of the mucus gel. To address this we have isolated mucins from normal, asthmatic and chronic bronchitic secretions. The asthmatic mucin reduced subunits were fractionated into four populations (I-IV) by anion-exchange HPLC. Amino acid and monosaccharide compositional analysis, as well as M(r) and size measurements, indicate that two of these populations (I and II) are glycoforms of the same or related apoprotein(s) and that the other populations contain two different apoproteins. A panel of antibodies and antisera recognizing the variable number tandem repeat (VNTR) of specific mucin apoproteins did not, as predicted, react with the glycosylated molecules, but after deglycosylation the majority of these probes (with the exception of those to MUC2, which were negative) reacted at a low level with each of the subunit populations. In contrast, an antiserum against a non-VNTR sequence of MUC5AC identified one of the populations (III) as the MUC5AC mucin. The MUC5AC reduced subunit had an M(r) of 2.2 x 10(6) and an RG (radius of gyration) of 57 nm. The genetic identities of the major mucin (populations I and II) and a minor component (population IV) were not established. The MUC5AC mucin was also identified as a major component in the pooled normal secretions from 20 individuals, whereas in a chronic bronchitic sample it was only a minor constituent. Furthermore, in all these different respiratory secretions the MUC5AC mucin appears as a similar biochemical entity, as assessed by Mono Q chromatography and agarose electrophoresis, suggesting that it may have a well-defined pattern of glycosylation in the respiratory tract.
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Perhaps the least understood aspect of polymer structure is the organization of backbone and side chains which prevails at the boundaries between contacting dissimilar phases. For example, the whole subject of biological adhesion—ranging from thrombus formation on implanted prosthetic materials through barnacle adhesion to ship bottoms—is limited by our lack of detailed knowledge of the predominantly proteinaceous “conditioning” films which adsorb on solid surfaces. These films are thought to be a prerequisite to cell adhesion (1,2). Cell membranes, themselves, are interfacial polymeric structures which are inadequately described despite intense, continuing study.
A chemically treated paper stick (Dextrostix) method of blood glucose estimation was compared to the alkaline potassium ferricyanide method in which an automated system (Auto Analyzer) of blood glucose estimation is used. Six medical technicians in a routine hospital laboratory performed the tests. Their instructions consisted solely of those accompanying the Dextrostix. Analysis of the data by the covariance technique suggests that, under the conditions of this study, the Dextrostix method has serious limitations. There was inconsistency, at least among technicians, in ability to discriminate shades of color, which rendered the Dextrostix method unsatisfactory for use as a mass-screening tool or as an alternate emergency laboratory procedure. It is also doubtful that the results of this method could be relied upon in the differential diagnosis of coma when the presence of hypoglycemia or hyperglycemia must be established with certainty.
The observed reduced adhesiveness of human intraoral mucosa, as compared with adjacent teeth, was determined for 14 healthy humans to correlate with differing measured intraoral contact angles for a variety of otherwise non-interacting test liquids on these two equally water-wettable surfaces under clinical conditions. Measurements were made on the front maxillary tooth surfaces and the-inner lower lip mucosal surfaces of the same subjects, either immediately upon first exposure of the oral tissues or after 30-60 s drying in ambient air. Lower critical surface tensions were found for wet mucosal surfaces (25.4 mN/m) than for wet tooth surfaces (31.2 mN/m). Exposure to air for up to 1 min increased the mucosal critical surface tensions by only 2 mN/m, and had even less influence on the measured properties of tooth surfaces. These data suggest that the observed 'bioabhesive' surface quality of oral mucosa, retaining negligible plaque, is associated with its critical surface tension in the zone near 25 mN/m, as found earlier for the natural fouling-resistant surfaces of dolphins and killer whales.
A salivary derived proteinaceous film will condition any surface exposed in the oral cavity prior to cell attachment. Studies have shown a strong correlation between the initial surface properties of a material and its cell retaining capacity, indicating that a mechanism may exist by which surface characteristics of a solid are transmitted through the adsorbed film. The aim of this study was to monitor the film organization of salivary secretions at defined liquid‐solid interfaces, and to evaluate the effect of the critical surface tension and the presence of microorganisms on the packing of the films. Fresh secretions from human parotid (HPS) and submandibular‐sublingual glands (HSMSL) were allowed to form thin films on surfaces of low and medium critical surface tensions under controlled flow conditions, in the presence and absence of microorganisms. Two strains of Streptococcus salivarius and Streptococcus sanguis were used as test microorganisms. The resulting films were monitored in situ by nondestructive surface analytical techniques, to avoid interference of either composition or organization of the adsorbed films. ATR infrared spectroscopy and ellipsometry were used to obtain information on the amount of protein and the thickness of the films. Both HPS and HSMSL formed films of approximately 200 Å thickness on surfaces of medium critical surface tension, but on the low energy surfaces HPS films were significantly thinner. More protein mass was detected in films on medium energy surfaces than on low energy surfaces. The presence of microorganisms during film deposition did not have a statistically significant effect on any of the parameters measured. These data indicate that the amount of salivary protein retained by a material is more directly related to critical surface qualities of the solid than to microorganisms in suspension, while the film's initial organization (e.g. packing, density) of salivary derived pellicles is also dependent on the specific protein‐surface combination.
Structures in human saliva were demonstrated using flash freezing, sectioning, fixing/staining and microscopic examinations. The microarchitecture and staining patterns of these structures and interactions with cellular material indicate the presence of the following salivary compartments: (1) a continuous water‐based electrolyte, (2) a continuous scaffold‐like network of amphiphilic macromolecules, (3) network cores with e.g. EGF/NGF‐activities, and (4) dispersed lipoid droplets. Through some acellular ingestion, networks and cores seem to engulf and pack objects like microorganisms and epithelial cells. This scavenging power and the ability of saliva to donate substances promoting wound healing provides an explanation for the instinctive sucking/licking of wounds observed in animals and humans.
The saliva in dry mouth subjects worked as well as that of the controls in providing lubricity. Their interaction with the saliva substitutes was similar to the behavior of that of the controls with the substitute. Based on the statistical analysis, it is likely that saliva in dry mouth subjects differs from that in normal healthy individuals in quantity alone and not quality.
An understanding of the basic structure, viscoelastic properties and interactions of mucin glycoproteins is of considerable interest to food science because of the important protective role that these macromolecules play in gastric physiology. The polymeric/colloidal behavior of mucins is complicated due to their large size (2–50 MDa) and complex structure with domains involving hydrophilic/hydrophobic, hydrogen bonds and electrostatic interactions, and their propensity to aggregate and form complexes with other polymers. These properties are also of direct relevance to the numerous diseases involving mucins, and to the problems of uptake of nutrients and delivering drugs through the mucus barrier. In this review we describe the current state of understanding of the relevant properties, with an emphasis on recent research. The findings described in this review are of direct relevance to the uptake of nutrients in digestion.
The cervical mucus plug is a large, complex structure within the cervical canal that is shed shortly before or during labor. We propose that the cervical mucus plug fulfills critical 'gate-keeper' functions based on its physical and immunologic properties, which help prevent ascending infection and preterm labor. The viscoelastic properties of the cervical mucus plug are determined by mucins (large glycoproteins), which can inhibit viral replication and exclude larger molecules and bacteria by preventing their diffusion through the plug. Furthermore, the innate and adaptive immunological properties of the cervical mucus plug are well suited for arresting bacterial infection by stimulating a robust inflammatory response. A possible association between an impaired gate-keeper function of the cervical mucus plug and preterm birth is discussed.
Conclusions regarding the physiological basis and disruptive effects of premenstrual symptoms may be biased because of the reliance on self-report questionnaires as a source of data. In order to examine this possible bias, women's perceptions of their cycle phase were separated experimentally from actual cycle phase. Women who were led to believe that they were premenstrual reported experiencing a significantly higher degree of several physical symptoms, such as water retention, than did women who were led to believe they were intermenstrual. Thus, because of these psychosocial influences on symptom reports, it seems necessary to reexamine previous conclusions regarding the magnitude of menstrual-related changes as well as their physiolocical basis.
Over the last fifteen years a detailed series of studies (Odeblad, 1973) has been undertaken which have utilized a number of different biophysical methods which include nuclear magnetic resonance (n.m.r.) techniques, viscometry, light scattering, sperm migration and tracer techniques involving exchange with heavy water (D2O, H217O). These have established a well defined hypothesis for the specific structure of cervical mucus in different endocrinal states in which there is an ordered arrangement of the mucin macromolecules which comprise the high viscosity component of cervical mucus, (Figure 1). In the ovulatory mucus these are arranged into bundles (“micelles”) with the low-viscosity aqueous phase or cervical plasma being present between the molecular bundles. These micelles are interconnected to form the gel network. The linking of the macromolecules is hormone dependent, due to hormone-regulated differences of the biosynthetic processes which are as yet undefined. The ovulatory micelle system (mucus type E) permits sperm propagation while the gestagenic mucus (type G) does not permit sperm entry or transmission through the genital tract. The mucin macromolecules which are synthesized during oestrogenic stimuli tend to crosslink in such a manner that a low-entropy system of elongated, branched molecular aggregates occur which is surrounded by its high water of hydration. The biosynthesis which occurs under the additional control of progestogens results in another type of cross-linking, in which there is a system of high entropy with the formation of far smaller micelles or not at all.
Samples of mixed saliva and of parotid and sublingual/submandibular saliva fractions from four donors were subjected to instantaneous solidification in liquid nitrogen followed by sectioning in a microtome/cryostat. The sections were stained with hematoxylin-eosin, periodic acid-Schiff, Alcian blue, Oil-red-O, or Sudan Black B and then examined at the light-microscopic level. In all the sectioned samples several previously never described features were observed, the most pronounced of which were a loose overall network structure and collections of lipoid droplets often in a loose arrangement. In the mixed saliva sample sections many of the microorganism-like structures were observed in large bunches associated with epithelial cells and densely staining saliva components. The present method was tested in a series of experiments for possible errors.
The endocervical canal is filled with a mucus gel, the properties of which vary during the ovulatory cycle. At mid-cycle the amount of mucus increases, mainly owing to an oestrogen-induced increased hydration of the gel, mucus becomes less visco-elastic and the penetration of the spermatozoa is facilitated. In contrast, under the influence of progesterone during the luteal phase, mucus turns into a less hydrated, highly visco-elastic structure which acts as a barrier to sperm. The mucus gel is formed by very large and structurally complex glycoproteins perfected by evolution to tease and disunite the scientists engaged in unravelling their secrets. The macromolecules are referred to as the mucus glycoproteins or the mucins. Hydrodynamic studies show that cervical mucus glycoproteins (Mr 10-15 x 10(6] behave as random coils, which occupy large spheroidal domains in dilute solution. The predicted 'linear' structure is supported by evidence obtained with electron microscopy. By this technique, the macromolecules are visualized as 'threads' with a skewed and polydisperse distribution of contour lengths (number-average length, 1.5 microns; range 0.5-5 microns). The macromolecules may be cleaved into subunits (Mr 2-3 x 10(6] by reduction of disulphine bonds and these fragments can be divided into large glycopeptides (T-domains; Mr 300,000-400,000) by trypsin. Most of the carbohydrate, which accounts for approximately 80% by weight of the macromolecule and occurs as a heterogeneous population of oligosaccharides, is enriched within the T-domains. The high-Mr glycopeptides thus correspond to long (of the order 100 nm) stretches of protein covered with 100-300 oligosaccharides which protect the core from proteolysis. These regions of the macromolecule are referred to as oligosaccharide 'clusters' and subunits of cervical mucins contain, on average, 3-5 of these 'clusters'. Each 'cluster' is flanked by stretches of protein which are less substituted with carbohydrate and, consequently, more sensitive to proteolysis. There is evidence that these parts of the core, referred to as the 'naked' regins, are folded and stabilized by disulphide bonds. Cervical mucus glycoproteins may thus be viewed as a linear array of oligosaccharide-rich 'clusters' alternating with structures reminiscent of a globular protein. Little is known about how the mucus glycoproteins interact to form the gel. The classical 'Odeblad concept' postulates that the mucins form bundles ('micelles') which are then interconnected in a hormone-dependent way. In contrast, light-scattering studies suggest that cervical mucus is an entangled net-work of long and flexible macromolecules.(ABSTRACT TRUNCATED AT 400 WORDS)
With foam components removed, mixed saliva from three donors were solidified in liquid nitrogen and sectioned, mounted, and fixed. Examination by transmission (TEM) and scanning (SEM) electron microscopy and energy-dispersive X-ray (EDAX) analysis were performed for paraformaldehyde-fixed sections, some of which were OsO4-postfixed. The TEM and certain SEM examinations showed the presence of fine and dense salivary network structures, seemingly originating from the major fibrous components. In OsO4-treated sections, TEM pictures showed reticulated arrangements with open cellular diameters down to 0.2 microns. The EDAX analyses particularly showed the presence of Ca, Fe, K, P, and S, with increased Ca readings in major components. Untreated sections showed that strands, with diameters of more than 1-2 microns, had more electron-dense central portions than peripheries and sometimes had interior, very electron-dense, granules. The observed features indicate that saliva has internal structures consistent with its colloid chemical characteristics.
CERTAIN changes in the physical properties of the cervical mucus are essential for the passage of sperms at the time of ovulation1. These changes are under hormonal control; oestrogens cause the mucus to be thin and clear whereas progestational agents render it thick, opaque, viscid and impenetrable to sperms2. The biochemical phenomena underlying these physical changes are not clear, but it is probable that quantitative or qualitative alterations in the glycoproteins and the smaller protein fractions are involved. The data on the changes in the protein composition of the cervical mucus during the menstrual cycle, however, are scanty and conflicting. Moghissi and Neuhaus3 have demonstrated changes in the protein fractions at the time of ovulation but these are inconsistent with the patterns described by Herve et al.4. These findings in turn differ, in spite of similar microimmuno-electrophoretic techniques being used from those of Schumacher et al.5 and from Trnka et al.6, who failed to demonstrate precipitable protein in non-pregnant mucus.
Chez les femmes la viscosité de la salive mise en relation avec la température du corps révèle le temps de l'ovulation aussi bien que les phases variées du cycle menstrual.
Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 X 10(6) and 5.9 X 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).
A retrospective study of 214 pregnancies in 100 consecutive patients with endometriosis revealed a significant increase (P<.01) in spontaneous abortions among women who conceived within 4 years prior to the diagnosis of endometriosis. A comparison of pregnancy outcome in a second group of 65 patients revealed a significant decrease (P<.01) in the spontaneous abortion rate following conservative surgery for endometriosis. Of 37 conceptions before surgery, 46% (17) were aborted spontaneously; after conservative surgery only 8% (4) of 50 conceptions terminated in abortion.
Cervical mucus is produced throughout the menstrual cycle. Sperm migration, however, is possible only during the periovulatory period of the cycle. Cervical mucus is also produced during the amenorrhoeic post-partum period. Post-partum mucus is very similar to luteal phase mucus except that it can allow sperm migration. In this study, mucus samples obtained from all these periods were classified according to their capacity to allow sperm migration. The biochemical characteristics of mucus samples that did (peri-ovulatory and 40% of post-partum samples) and did not (luteal and 60% of post-partum samples) allow sperm migration were then compared. Mucus samples with positive sperm migration showed the highest percentage of water and lowest protein and glycoprotein concentration (per ml of mucus). In addition, post-partum mucus samples with positive sperm migration showed lower concentrations of proteins and glycoproteins than post-partum mucus samples that did not allow sperm migration. However, the amount of glycoproteins per mg of protein was similar between post-partum samples that were positive and negative for sperm migration. These data suggest that the carbohydrate composition of the glycoproteins is playing a key role in the ability of cervical mucus to accept spermatozoa.
We summarize the epidemiologic literature on the effect of life-style factors such as cigarette smoking, alcohol and caffeine consumption, physical exercise, body mass index, and drug use on female infertility. We identified relevant papers through MEDLINE, Index Medicus, and a manual review of reference lists. Risk factors that affect the risk of primary tubal infertility and that were corroborated in two or more studies include use of intrauterine devices (especially the Dalkon Shield) and cigarette smoking. We identified extremes in body size as a risk factor for primary ovulatory infertility. Cocaine, marijuana and alcohol use, exercise, caffeine consumption, and ever-use of thyroid medications were possible risk factors for various subtypes of primary infertility. Few risk factors have been assessed or identified for secondary infertility or other less common subtypes, such as cervical or endometriosis-related infertility.
Previous studies have shown that peptides derived from the N-terminal region of the low molecular mass human salivary mucin, MUC7, possess potent in vitro cidal activity against Candida albicans and other medically important fungi. MUC7 12-mer (residues 40-51 of the parent MUC7) peptide, having the optimal size and a net charge of +6, was found to be anticandidal in human saliva (clarified and unclarified), and its candidacidal potency was found to be superior to that of histatin 5 12-mer (Hsn5 12-mer). We have, therefore, explored the candidacidal potency of MUC7 12-mer (l and d isomers) and Hsn5 12-mer peptides in vivo. In vitro killing assay was performed to establish killing activity of the peptides against C. albicans prior to in vivo experiments. A murine model of oral candidiasis that has the characteristics of oral thrush in humans was employed for the in vivo studies, based on a previous protocol. Upon candidal induction, antifungal treatment application using agents emulsified in Pluronic F127 was performed for six consecutive days. Amphotericin B and clotrimazole emulsified in the same delivery system were used as positive control drugs. Candidacidal efficacy was evaluated microbiologically and histopathologically. Results demonstrated a considerable reduction of fungal burden by the MUC7 12-mer peptides (l and d), comparable to control drugs, and this effect was statistically significant, unlike the effect seen with Hsn5 12-mer. Murine oral candidiasis model employed in this study is suitable to test the candidacidal agents employing Pluronic F127. In conclusion, MUC7 12-mer appears to be a promising candidate as an antifungal agent for oral candidiasis.
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