Cementum structure in Beluga whale teeth.

A large fraction of the volume of Beluga whale (Delphinapterus leucas) teeth consists of cementum, a mineralized tissue which grows throughout the life of the animal and to which the periodontal ligaments attach. Annular growth bands or growth layer groups (GLGs) form within Beluga cementum, and this study investigates GLG structure using X-ray fluorescence mapping and X-ray diffraction mapping with microbeams of synchrotron radiation. The Ca and Zn fluorescent intensities and carbonated hydroxyapatite (cAp) diffracted intensities rise and fall together and match the light-dark bands visible in transmitted light micrographs. Within the bands of maximum Ca and Zn intensity, the ratio of Zn to Ca is slightly higher than in the minima bands. Further, the GLG cAp, Ca and Zn modulation is preserved throughout the cementum for durations >25year. STATEMENT OF SIGNIFICANCE: Cementum is an important tooth tissue to which the periodontal ligaments attach and consists primarily of carbonated apatite mineral and collagen. In optical microscopy of cementum thin sections, light/dark bands are formed annually, and age at death is determined by counting these bands. We employ synchrotron X-ray diffraction and X-ray fluorescence mapping to show the bands in Beluga whale cementum result from differences in mineral content and not from differences in collagen orientation as was concluded by others. Variation in Zn fluorescent intensity was found to be very sensitive indicator of changing biomineralization and suggest that Zn plays an important role this process.

Bovine and equine peritubular and intertubular dentin.

Dentin contains 1-2µm diameter tubules extending from the pulp cavity to near the junction with enamel. Peritubular dentin (PTD) borders the tubule lumens and is surrounded by intertubular dentin (ITD). Differences in PTD and ITD composition and microstructure remain poorly understood. Here, a (∼200nm)(2), 10.1keV synchrotron X-ray beam maps X-ray fluorescence and X-ray diffraction simultaneously around tubules in 15-30µm thick bovine and equine specimens. Increased Ca fluorescence surrounding tubule lumens confirms that PTD is present, and the relative intensities in PTD and ITD correspond to carbonated apatite (cAp) volume fraction of ∼0.8 in PTD vs. 0.65 assumed for ITD. In the PTD near the lumen edges, Zn intensity is strongly peaked, corresponding to a Zn content of ∼0.9mgg(-1) for an assumed concentration of ∼0.4mgg(-1) for ITD. In the equine specimen, the Zn K-edge position indicates that Zn(2+) is present, similar to bovine dentin (Deymier-Black et al., 2013), and the above edge structure is consistent with spectra from macromolecules related to biomineralization. Transmission X-ray diffraction shows only cAp, and the 00.2 diffraction peak (Miller-Bravais indices) width is constant from ITD to the lumen edge. The cAp 00.2 average preferred orientation is axisymmetric (about the tubule axis) in both bovine and equine dentin, and the axisymmetric preferred orientation continues from ITD through the PTD to the tubule lumen. These data indicate that cAp structure does not vary from PTD to ITD.

Near tubule and intertubular bovine dentin mapped at the 250 nm level.

In this study, simultaneous diffraction and fluorescence mapping with a (250nm)(2), 10.1keV synchrotron X-ray beam investigated the spatial distribution of carbonated apatite (cAp) mineral and elemental Ca (and other cations including Zn) around dentin tubules. In 1µm thick sections of near-pulp root dentin, where peritubular dentin (PTD) is newly forming, high concentrations of Zn, relative to those in intertubular dentin (ITD), were observed adjacent to and surrounding the tubule lumens. Some but not all tubules exhibited hypercalcified collars (high Ca signal relative to the surrounding ITD), and, when present, the zone of high Ca did not extend around the tubule. Diffraction rings from cAp 00.2 and 11.2+21.1+30.0 reflections were observed, and cAp was the only crystal phase detected. Profiles of Ca, Zn and cAp diffracted intensities showed the same transitions from solid to tubule lumen, indicating the same cAp content and organization in ITD far from the tubules and adjacent to them. Further, the matching Ca and diffraction profiles demonstrated that all of the Ca is in cAp or that any noncrystalline Ca was uniformly distributed throughout the dentin. Variation of 00.2 and 11.2+21.1+30.0 diffracted intensity was consistent with the expected biaxial crystallographic texture. Extension of X-ray mapping from near 1µm resolution to the 250nm level, performed here for dentin and its tubules, will provide new understanding of other mineralized tissues.

Regulation of vocal fold transepithelial water fluxes.

Vocal fold hydration is critical to phonation. We hypothesized that the vocal fold generates bidirectional water fluxes, which are regulated by activity of the Na(+)-K(+)- ATPase. Western blots and immunohistochemistry demonstrated the presence of the alpha-subunit Na(+)-K(+)-ATPase in the canine vocal fold (n = 11). Luminal cells, basal and adjacent one to two layers of suprabasal cells within stratified squamous epithelium, were immunopositive, as well as basolateral membranes of submucosal seromucous glands underlying transitional epithelia. Canine (n = 6) and ovine (n = 14) vocal fold mucosae exhibited transepithelial potential differences of 8.1 +/- 2.8 and 9.3 +/- 1.3 mV (lumen negative), respectively. The potential difference and short-circuit current (ovine = 31 +/- 4 microA/cm(2); canine = 41 +/- 10 microA/cm(2)) were substantially reduced by luminal administration of 75 microM acetylstrophanthidin (P < 0.05). Ovine (n = 7) transepithelial water fluxes decreased from 5.1 +/- 0.3 to 4.3 +/- 0.3 microl x min(-1) x cm(-2) from the basal to luminal chamber and from 5.2 +/- 0.2 to 3.9 +/- 0.3 microl x min(-1) x cm(-2) from the luminal to basal chamber by luminal acetylstrophanthidin (P < 0.05). The presence of the Na(+)-K(+)-ATPase in the vocal fold epithelium and the electrolyte transport derived from its activity provide the intrinsic mechanisms to regulate cell volume as well as vocal fold hydration.

Development of a measure of attitude toward nutrition in patient care.

BACKGROUND: Development of reliable measures of medical student and resident attitudes about nutrition in patient care is needed before the effects of educational interventions or clinical experience can be gauged. This report describes the systematic development of a measure of attitude toward nutrition in patient care. It presents evidence about scale reliability and the absence of response bias that endorses the trustworthiness of data from the measure. METHODS: An eight-step attitude scale development procedure was used to create the Nutrition In Patient care Survey (NIPS). Data from five samples of first- and second-year medical students and first-year medical residents were subjected to factor analysis (PA2, varimax rotation), reliability analyses, and statistical analyses to test for demographic bias in the attitude data. RESULTS: A 45-item attitude measure was developed that contains five subscales derived from the factor analysis: (1) nutrition in routine care (NRC, 8 items); (2) clinical behavior (CB, 20 items); (3) physician-patient relationship (PPR, 8 items); (4) patient behavior/motivation (PBM, 3 items); and (5) physician efficacy (PE, 6 items). Each subscale yields reliable data in terms of internal consistency (alpha coefficients) and stability (test-retest reliability). Medical student and resident demographic variables have negligible influence on attitude scores. DISCUSSION: The NIPS subscales yield reliable data that can be used to assess outcomes in evaluation research on educational or clinical interventions or to predict patient care practices. Systematic attitude scale development increases the likelihood that the resulting measures will produce useful, trustworthy data.

Expression of retinoic acid receptors in non-neoplastic epithelial disorders of the vulva and normal vulvar skin.

Retinoids and their nuclear retinoic receptors (RARs) are important modulators of epidermal cell proliferation and terminal differentiation. Aberrant expression of RARs in the epidermis has been found to be associated with altered differentiation capacity of keratinocytes. In this study, the expression of the various types of RARs (RAR-alpha, RAR-beta, and RAR-gamma) was investigated in surgical specimens from 17 patients with vulvar lichen sclerosus, 12 patients with vulvar squamous cell hyperplasia, and 11 specimens of normal vulvar skin by nonradioactive in situ hybridization. The results demonstrate that RAR-alpha expression is significantly decreased in lichen sclerosus (p < 0.0001) and squamous cell hyperplasia (p = 0.007) compared with normal vulvar skin. Furthermore, in normal vulvar skin RAR-alpha mRNA is mainly located in the suprabasal epidermal cell layers, whereas in lichen sclerosus RAR-alpha is expressed predominantly in the basal cell layers. In squamous cell hyperplasia RAR-alpha expression occurs in all cell layers. Compared with normal vulvar skin, RAR-gamma expression is higher in lichen sclerosus (p = 0.026), but no statistically significant differences are seen in squamous cell hyperplasia. These results suggest that partial loss and abnormal localization of RAR-alpha expression as well as increased RAR-gamma expression may play a role in the etiology of non-neoplastic epithelial disorders of the vulva.

In situ localization of S1-labeled actin filaments in chick lens epithelial cells.

The actin filaments in the lens epithelial cells of three-day and eight-day post-hatched chicks have been labeled in situ with myosin subfragment 1 (S1). Labeling was accomplished by injuring the lens transcorneally with an ultramicroneedle 5 min or 24 hr before detergent treatment and incubation in S1. A band of filaments found at the epithelio-fiber junction in normal, uninjured chick lens is labeled in the 5 min and 24 hr injury. A subcapsular labeled band is found only in the 24 hr injury, and may be the result of a healing process.

Exogenous hyaluronidases and degradation of hyaluronic acid in the rabbit eye.

The infusion of two enzymes that degrade hyaluronic acid--testicular hyaluronidase and Streptomyces hyaluronidase--was evaluated by quantitative aqueous perfusion of rabbit eyes and by analyses of glycosaminoglycans (GAGs) isolated from the enzyme-treated eyes. The infusion of 1 and 10 units of Streptomyces hyaluronidase (SH) was considerably more effective than the infusion of 10 and 100 units of testicular hyaluronidase (TH) in reducing aqueous outflow resistance. As a result of the infusion of heat-inactivated enzymes, only a moderate decrease of hyaluronic acid in the aqueous outflow pathway was observed. There was no significant "wash-out" of other GAG material, ie, keratan sulfate, heparan sulfate, and hybrid dermatan sulfate-chondroitin sulfate. The SH enzyme, tested by infusion and isolation of GAGs or by in vitro analyses of the rate and extent of degradation of GAGs, completely removed all hyaluronic acid and did not alter the other GAGs. In contrast, the TH enzyme was only partially effective in degrading susceptible GAGs. The results of these studies indicate that SH is more effective than TH in decreasing aqueous outflow resistance and that hyaluronic acid is an important GAG contributor to aqueous outflow resistance in the normal rabbit eye.

A sodium dodecyl sulfate--polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000.

A discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described which provides superior resolution of polypeptides with molecular weights from approximately 2500 to 90,000. The system utilizes a relatively low-mobility acetate ion in the stacking gel and high-mobility strong anions, sulfate and chloride, as leading and trailing ions in the separating gel. The entire system is run at pH 7.8. The separating gel contains 8 M urea, and can be used at acrylamide concentrations from 5 to 18%, all with 5% crosslinker concentrations. Using a number of protein standards, the calibration curves obtained with this system are linear over the molecular weight range from 2500 to 90,000, regardless of acrylamide concentration. These studies indicate that by providing good resolution of small peptides, this system greatly extends the utility of one-dimensional peptide mapping techniques.

A low-molecular-weight soluble protein from bovine lingual epithelium. II. Purification and characterization.

In an earlier study of rat lingual epithelium, we examined the total SDS-soluble protein from epithelia isolated at different states of development by polyacrylamide gel electrophoresis. In this study, rather than work with total SDS-soluble proteins, we have carried out a series of sequential extractions of bovine lingual epithelium. To separate epithelium from connective tissue, slices of dorsal tongue mucosa were incubated in a solution containing EDTA and 4 proteolytic enzyme inhibitors. We have isolated and partially characterized a low-molecular-weight (LMW) protein from the phosphate-buffered saline extract of bovine lingual epithelium. In the work reported here, we describe some of the biochemical and immunologic characteristics of this protein. The bovine lingual LMW protein has a molecular weight of 8700 +/- 450, an isoelectric point of 4.7 +/- 0.2 pH units, and a high content of the acidic amino acids aspartate and glutamate. We prepared an antibody to LMW protein and examined its specificity by a microenzyme-linked immunosorbant assay (ELISA). We found that the antibody to LMW protein reacts very strongly against LMW protein while it exhibits no cross-reactivity with low levels of an authentic keratin protein but moderate cross-reactivity at higher concentrations of this authentic keratin protein. In a previous publication we have reported the immunohistochemical localization and distribution of this LMW protein.

Immunohistochemical localization of a low molecular weight, soluble protein from bovine lingual epithelium.

A low molecular weight (LMW) protein was isolated from bovine tongue epithelium and an antiserum to this protein elicited in rabbits. The indirect peroxidase-antiperoxidase (PAP) technique was used to localize LMW protein in several tissues from six mammalian species: cow, rat, mouse, squirrel, rabbit, and man. Immunoreactivity was demonstrable in stratified squamous epithelia from skin, tongue, cheek, esophagus, vagina, and palate. Epidermal derivatives, such as hair follicles, sebaceous glands and ducts of certain glands were also positively stained. Cornea exhibited weak immunoreactivity as did rabbit bladder. Other types of epithelia including those seen in kidney, thyroid, intestine, trachea, liver, submandibular gland, pancreas and uterus, were not immunoreactive when tested with antiserum to LMW protein. The antiserum was rendered unreactive after absorption with LMW protein but, when absorbed with a keratin polypeptide, most of the immunoreactivity was preserved. It is concluded that the distribution of the soluble LMW protein is similar to that of the insoluble keratin proteins in stratified squamous epithelia but the former is not demonstrable in many simple epithelia that contain keratins.

A specific stain for sulfhydryl groups in proteins after separation by polyacrylamide gel electrophoresis.

A new method is described for specifically staining protein sulfhydryl groups after the proteins have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in slab gels. The stain will detect as little as 0.25 microgram of lysozyme and 1 microgram of most other proteins; the range of sensitivity for a specific protein depending on its sulfhydryl content. Proteins with no cysteine residues (type I collagen) and glycoproteins do not cause spurious staining.

Diminished axonal transport of glycoproteins in the senescent rat brain.

At various time intervals (10, 15, 20, 25, 30 min) after injection of 3H-fucose into the medial septal nucleus of young adult (3 months old) and senescent (25 months old) Fischer-344 rats, the specific activities of trichloroacetic acid-phosphotungstic acid (TCA-PTA) soluble and insoluble fractions were determined in the medial area of the septum and in three successive rostro-caudal sections of the hippocampal formation containing mainly the dentate gyrus, but also its hilus with fields CA4 and CA3c of the hippocampus. The rate of 3H-fucose incorporation into glycoproteins of the septum did not differ in young adult and senescent rats. Part of the TCA-PTA soluble and insoluble radioactive material was transported through the septo-hippocampal pathway to the dentate gyrus. This transport was inhibited by the injection of colchicine into the septum prior to 3H-fucose injection and was completely blocked by electrolytic lesion of the medial septal nucleus. The arrival time and the amount of the TCA-PTA soluble radioactive material transported to the dentate gyrus did not differ in young adult and senescent rats. However, the TCA-PTA insoluble labelled glycoprotein was transported to the dentate gyrus in a significantly smaller amount and during a longer period of time in the senescent animals. This age-related change may reflect a reduction in amount and/or in rate of axonal transport of glycoproteins in the septo-hippocampal pathway of senescent rats.

Developmental changes in the protein profile of cornifying lingual epithelium.

Epithelia from the tongue dorsum of 14- to 21-day embryos, 21-day embryos, 3-week-old, and adult rats were separated from their connective tissues by incubation in balanced salt solution containing EDTA. Aliquots from total extracts of these tissues were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Scans of gels stained with fast green (FG) revealed more than 20 peaks. Ten major peaks ranging from apparent molecular weights (MW) of 120,000 to 14,000 daltons comprised about 70% of the total protein on each gel. This report focuses primarily on two pairs of peaks, arbitrarily numbered 2 and 3 (MWs 71,000 and 69,000) and 9 and 10 (MWs 17,500 and 14,000). Peaks 9 and 10 predominated in the 15-day embryos where they comprised about 30% of the total protein. As development proceeded, there was a gradual shift in the protein profile in favor of peaks 2 and 3 until on the 20th day the relative amounts of these peaks reached a maximum and peaks 9 and 10 decreased in relative amounts. The protein profile on the 20th fetal day resembled that of the 3-week-old rats and the adults. The rise in the relative amounts of peaks 2 and 3 coincided with the morphologic appearance of large numbers of tonofilaments and the onset of cornification. When the gel was stained by a procedure specific for sulfhydryl groups, peaks 9 and 10 were especially reactive after the 18th day; plainmetric analysis revealed that these had twice the relative affinity for this stain than for FG whereas other peaks had equal or less affinity. The incorporation of [3H]cystine into peaks 9 and 10 was relatively greater than into the other proteins.

Cultured neuroblastoma cells and halothane: effects on cell growth and macromolecular synthesis.

Cultured mouse neuroblastoma cells were grown in air-CO2 or air-CO2-halothane-gassed incubators. In the presence of halothane the growth rate of the cells was inhibited in a dose-dependent manner; 2 per cent halothane completely inhibited cell growth, while at 0.3 per cent halothane, the growth rate was 74 per cent of the control rate. The biosynthesis of protein and RNA in cells grown in the control atmosphere and that in cells grown in 1 per cent halothane were compared by several techniques. No significant difference between the rates of synthesis of these two macromolecules could be detected. Furthermore, a comparison of labeled protein and RNA by SDS-polyacrylamide gel electrophoresis revealed no qualitative difference. From this and previous work it is concluded that halothane affects the morphology and growth rate of cultured mouse neuroblastoma cells by disrupting cytoplasmic actin-like micro-filaments.