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Biomedical subjects

A R Hand

Publications and source records attributed to A R Hand.

At least 91 records · Page 5Linked to original sources
PubMed1988

Localization and enumeration of fimbria-associated adhesins of Bacteroides loescheii.

Monoclonal antibodies that specifically inhibit coaggregation between Bacteroides loescheii PK1295 and its two gram-positive partners Streptococcus sanguis 34 and Actinomyces israelii PK14 were used to enumerate and localize two distinct types of fimbria-associated adhesins on the surface of B. loescheii. Binding studies with radiolabeled monoclonal antibodies indicated that a maximum (Bmax calculated from Scatchard plots) of approximately 400 adhesin molecules specific for S. sanguis and 310 adhesin molecules specific for A. israelii reside on the surface of the cell. Immunoelectron microscopy revealed that the adhesins were not an integral part of the fimbrial subunit; rather, they were usually found on the distal portion of the structures arranged in a random fashion.

Antibodies, Monoclonal↗
PubMed1987

Endocytosis of native and cationized ferritin by intralobular duct cells of the rat parotid gland.

The ability of the intralobular ducts of the rat parotid gland to take up protein from the lumen was examined after retrograde infusion of native and cationized ferritin. At high concentrations (3-10 mg/ml), cells of both intercalated- and striated ducts avidly internalized the tracers. No differences were noted in the mode of uptake or fate of native or cationized ferritin. Large, apical ferritin-containing vacuoles up to 5 microns in size were present in cells of the intercalated ducts after infusion for 15 min. Small, smooth-surfaced spherical or flattened vesicles and tubules containing ferritin were also observed, often in association with the large vacuoles. Ferritin uptake increased with increasing infusion time, up to 1 h. Uptake by the striated ducts was less consistent than by the intercalated ducts, and occurred mainly in small vesicles and tubules. Secondary lysosomes became labeled with ferritin in both cell types. Ferritin was not observed in the Golgi saccules, nor was it discharged from the cells at the basolateral surfaces. At low concentrations (0.3-1 mg/ml), uptake was reduced, especially by cells of intercalated ducts, and differences were noted in the behavior of the two tracers. Cationized ferritin was internalized mainly into vesicles and tubules of cells of striated ducts; little uptake of native ferritin occurred at low concentrations. These results demonstrate that the ductal cells of the salivary glands are capable of luminal endocytosis of foreign proteins. They also suggest that in addition to modifying the primary saliva by electrolyte reabsorption and secretion, and secretion of various glycoproteins, the ductal cells are able to reabsorb proteins secreted by the acinar cells.

Animals↗
PubMed1987

Fimbria-associated proteins of Bacteroides loescheii PK1295 mediate intergeneric coaggregations.

Bacteroides loescheii PK1295 serves as a coaggregation bridge between Streptococcus sanguis 34 and Actinomyces israelii PK14, two gram-positive oral bacteria that are otherwise unable to coaggregate. Whereas coaggregation with S. sanguis 34 is inhibited by lactose, no simple sugar was found that inhibited coaggregation with A. israelii PK14. Coaggregation-defective (Cog-) mutants of B. loescheii PK1295 were isolated for the purpose of identifying the surface components responsible for the interaction with each coaggregation partner. Selection for spontaneously occurring Cog- mutants gave rise to two phenotypic classes of mutants. Type I lost the ability to coaggregate with S. sanguis 34, whereas type II failed to coaggregate with either S. sanguis 34 or A. israelii PK14. Purified fimbriae from the parent agglutinated cells of both partners, and agglutination with S. sanguis 34 was inhibited by lactose. Denaturing polyacrylamide gel electrophoresis and immunoblot analysis demonstrated the presence of both a 75- and a 43-kilodalton (kDa) protein associated with parental fimbriae, but only a 43-kDa protein was seen with fimbriae prepared from the type I mutant. Neither polypeptide was found in similar preparations from the type II mutants. Our data suggest that coaggregation of B. loescheii PK1295 with both gram-positive partners is mediated by fimbria-associated proteins present on the surface of the gram-negative organism and that the 75- and 43-kDa polypeptides are responsible for the recognition of S. sanguis 34 and A. israelii PK14 cells, respectively.

Actinomyces↗
PubMed1987

Secretion of lingual lipase and amylase from rat lingual serous glands.

The effects of various secretagogues on the release of lingual lipase and amylase from rat lingual serous glands was examined in vitro and in vivo. After incubation, the media and tissues were assayed for lingual lipase and amylase activity to determine percent of secretion. In vitro secretion of lingual lipase and amylase stimulated by the cholinergic agonist, carbamylcholine chloride (carbachol), was 28.3 +/- 1.7, 48.0 +/- 3.2, and 55.9 +/- 2.4% and 18.1 +/- 1.7, 26.4 +/- 3.0, and 28.0 +/- 2.5%, respectively, for 30-, 60-, and 90-min incubations. The beta-adrenergic agonist, isoproterenol, and the adenylate cyclase activator, forskolin, elicited very little secretion in vitro; the 90-min values with isoproterenol were 16.8 +/- 7.1% lingual lipase and 6.0 +/- 2.6% amylase. The alpha-adrenergic agonist, phenylephrine, did not stimulate enzyme secretion. Morphological assessment of incubated tissues revealed that carbachol induced a rapid and extensive degranulation of the acinar cells, while isoproterenol caused only minimal exocytosis. In vivo stimulation by the cholinergic agonist, pilocarpine, caused rapid secretion, with maximal secretion occurring by 1 h. In vivo secretion stimulated by isoproterenol was slow, but by 4 h secretion was comparable to that induced by pilocarpine. In vitro, there was a significant difference between the percentages of lingual lipase and amylase secreted, which could not be accounted for by the presence of proteases or microbial products or the lack of stability of the enzymes during the incubation period. Neurotransmitter regulation of protein secretion by the lingual serous (minor salivary) glands appears to be principally cholinergic in contrast to the beta-adrenergic stimulation of protein secretion by the parotid (major salivary) gland.

Adrenergic alpha-Agonists↗
PubMed1987

Salivary gland ultrastructure and cyclic AMP-dependent reactions in Spacelab 3 rats.

Environmental stimuli influencing catecholamine levels induce changes in cyclic AMP-dependent reactions and cell morphology in the rat parotid. Responses of salivary glands to spaceflight were determined by measurement of cyclic AMP-mediated reactions in fresh-frozen salivary glands and by microscopic evaluation of ultrastructure in fixed parotid glands. Decreased cell-free protein phosphorylation occurred in parotid glands in three of five flight animals. Protein kinase activity ratios were decreased in the soluble and increased in the particulate fractions of Spacelab 3 (SL-3) rat sublingual glands, compared with ground controls. Biochemical analyses show that effects of space flight on salivary glands are similar to those induced experimentally by physiological manipulation or alteration of catecholamine levels. Morphological evaluation of three SL-3 rat parotid glands showed increased numbers of lysosomes, autophagic vacuoles containing degenerating secretory product, and accumulation of lipid droplets. Since these animals lost weight, consistent with disruption of food and water consumption, morphological changes may in part be due to decreased masticatory stimulation, as occurs with reduced food intake or a liquid diet. The observed changes may reflect physiological responses of the gastrointestinal and autonomic systems to effects of spaceflight.

Animals↗
PubMed1987

Endocytosis of proteins by salivary gland duct cells.

The ability of the intralobular duct cells of the rat parotid gland to take up protein from the lumen was examined by retrograde infusion of exogenous proteins and by immunogold localization of endogenous secretory proteins. Small amounts of native horseradish peroxidase (HRP) were taken up by intercalated and striated duct cells, and were present in small vesicles, multivesicular bodies, and lysosomes. In contrast, HRP modified by periodate oxidation was avidly internalized by the duct cells and was present in large apical vacuoles that acquired lysosomal hydrolase activity. Native and cationized ferritin were taken up in a similar manner when infused at a high concentration (up to 10 mg/mL). At lower concentrations (0.3-1.0 mg/mL), endocytosis of cationized ferritin occurred mainly in small apical tubules and vesicles in striated duct cells. Little native ferritin was taken up at these concentrations. After stimulation of acinar cell secretion by isoproterenol, similar vacuoles were occasionally observed in both intercalated and striated duct cells. Labeling of thin sections with antibodies to amylase and to a 26,000-dalton secretory protein (protein B1), followed by protein A-gold, revealed the presence of these proteins in the vacuoles, indicating endocytosis of acinar secretory proteins by the duct cells. Although uptake of acinar proteins by duct cells occurs at a low rate in normal animals, previous work suggests that extensive endocytosis may occur in certain pathological conditions. This may be a mechanism for removing abnormal or modified proteins from saliva before it reaches the oral cavity.

Animals↗
PubMed1987

Further characterisation of the extracellular matrix in the mandibular condyle in neonatal mice.

This study provides newer information concerning the extracellular matrix of neonatal condylar cartilage--a genuine representative of a secondary type of cartilage. In addition, the data presented hereby indicate that the condylar cartilage contains a population of progenitor cells that synthesise Type I collagen rather than Type II. Under normal conditions in vivo local biomechanical factors influence the progenitor cells to differentiate into cartilage cells and thereby to shift their synthetic pathway from Type I collagen to Type II collagen--the typical collagen of cartilage extracellular matrix. In the absence of such biomechanical effects the condylar progenitor cells seem to proceed with their inherent differentiation pathway and express an osteogenic phenotype (Fig. 21).

Animals↗
PubMed1987

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles.

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.

Animals↗
PubMed1987

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells.

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.

Animals↗
PubMed1986

T lymphocyte-dependent evolution of bacterial cell wall-induced hepatic granulomas.

Injection of streptococcal cell walls (SCW) i.p. into susceptible rats results in dissemination of SCW primarily to the liver, spleen, bone marrow, and peripheral joints. Within the liver, the SCW are phagocytized by the Kupffer cells, initiating a sequence of events leading to the formation of hepatic granulomas. The granulomas are characterized by large numbers of W3/13+, W3/25+ T lymphocytes and Ia+, esterase-positive macrophages. The generation of inflammatory mediators by these mononuclear cells appears to be central to the evolution of the granulomas and the subsequent fibrotic sequelae evoked by the SCW. In the absence of functional T lymphocytes (athymic rats), injection of SCW does not trigger lymphokine production, and organized granulomas do not develop in the livers. Furthermore, inhibition of T lymphocyte proliferation and lymphokine synthesis pharmacologically by cyclosporin A administration in euthymic animals inhibits SCW-induced hepatic granuloma development. Although macrophage function is apparently not impaired as evidenced by IL 1 and PGE2 production, a chronic inflammatory response to SCW cannot be sustained in the absence of T lymphocyte participation. These studies provide insight into the cellular and molecular mechanisms leading to formation and maintenance of chronic granulomatous lesions.

Animals↗
PubMed1986

Bacterial cell wall-induced hepatic granulomas. An in vivo model of T cell-dependent fibrosis.

In vitro studies implicate a molecular link between inflammatory mononuclear cells and alterations in fibroblast growth and function. We have extended these observations in an experimental animal model in which we document the T cell-dependence of fibrosis that occurs after activation of the cell-mediated immune system by specific antigen. Chronic granulomatous lesions were induced in the livers of susceptible rats by the intraperitoneal injection of group A streptococcal cell walls (SCW). The development of granulomas that are composed primarily of lymphocytes and macrophages was associated with the recruitment and proliferation of connective tissue cells. Furthermore, this expanded population of fibroblasts generated a collagenous structure consisting primarily of types I and III collagen around the granuloma. The progression of these chronic inflammatory lesions leads to the formation of fibrotic nodules throughout the livers of the treated animals. Intact granulomas, as well as mononuclear cells derived from the granulomas, spontaneously elaborated a soluble factor(s) that stimulates fibroblast proliferation. Physicochemical analysis revealed that the primary granuloma-derived peak of fibroblast growth activity corresponded to an apparent Mr of 40,000, which is consistent with a previously described T lymphocyte--derived fibroblast-activating factor (FAF) in guinea pig and human. Furthermore, the fibrosis that occurs in the granuloma is apparently T cell--dependent, since no fibrotic lesions developed in SCW-injected athymic nude rats nor in SCW-injected animals treated with the T cell inhibitor, cyclosporin A (CsA). Mononuclear cells from neither of these functionally T cell--deficient animals could generate FAF activity. These data show a role for T lymphocyte--derived cytokines in the development of hepatic fibrosis in SCW-injected rats.

Animals↗
PubMed1986

Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo.

The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulum-lysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors.

Acid Anhydride Hydrolases↗
PubMed1986

Exocytosis in rat parotid acini after in vitro treatment with forskolin is accompanied by cellular redistribution of regulatory subunits of cyclic AMP-dependent protein kinase.

Incubation of rat parotid acinar tissue with 1 mumol/L forskolin resulted in progressive exocytosis which was virtually complete after a 30-minute incubation period. Cyclic AMP binding to protein kinase (cA-PK) regulatory (R) subunits, determined by photo-affinity labeling with [32P]-8-azido cyclic AMP, was found to increase in a time-dependent manner in the 10,000-g supernatant fraction of a broken cell preparation. An apparent redistribution of protein kinase R subunits took place in the 600-g supernatant after in vitro treatment of cells with forskolin. In control cells, RI and RII subunits and a 35-to-40-kdal fragment were present in approximately equal amounts throughout the incubation. Azido labeling of RII appeared either to increase or to remain unchanged, while that of RI decreased in the 600-g pellet. Only type I isozyme R subunits were found in the 600-g pellet in either the absence or presence of forskolin. These finding indicate that a temporal relationship exists between stimulated protein exocytosis and cyclic AMP-dependent protein kinase activation. Forskolin stimulation of adenylate cyclase in salivary gland cells therefore provides a defined system for the study of cyclic AMP-mediated protein secretion.

Animals↗
PubMed1985

Mitosis and hypertrophy of intercalated duct cells and endothelial cells in the isoproterenol-treated rat parotid gland.

Chronic treatment of rats with isoproterenol (IPR) induces mitosis and differentiation of intercalated duct cells, and mitosis and hypertrophy of endothelial cells of the microvasculature in the parotid gland. Mitoses occurred in duct cells situated at or near the acinar-intercalated duct junction, and were most numerous after six IPR injections. These cells increased in size, contained numerous electron-lucent granules, and were incorporated into the hypertrophic acini. The granules contained branching filamentous or finely reticulated material, and did not react with histochemical stains for carbohydrate. Capillary and venule endothelial cell mitoses were frequently seen after four to six IPR injections. The endothelial cells increased in thickness, contained abundant cytoplasmic organelles, lacked fenestrations, and had few pinocytotic vesicles. These results demonstrate the extensive nature of the response to chronic IPR treatment, and are consistent with the concept that the intercalated ducts have an important role in salivary gland physiology.

Animals↗
PubMed1985

Horseradish peroxidase: factors affecting its distribution after retrograde infusion into the rat parotid gland.

Previous studies have shown that tight junctions of the unstimulated rat parotid gland are impermeable to retrogradely administered tracers such as myoglobin. Permeability is increased following beta-adrenergic stimulation, allowing the tracers to reach the intercellular and interstitial spaces. Reaction product of retrogradely infused horseradish peroxidase (HRP) and lactoperoxidase in found in the intercellular and interstitial spaces in both resting and stimulated glands, and many acinar and duct cells contain diffuse cytoplasmic reaction product. In this study we investigate several factors that might influence the distribution of HRP in the parotid gland. Tracer distribution was similar with HRP obtained from different suppliers, with different HRP preparations (Sigma types II, VI, VIII, and IX), and at HRP concentrations of 0.1-10 mg/ml. Inclusion of various saccharides in the infusion solution had no effect on the distribution of reaction product. Inhibition of the enzymatic activity of HRP by extraction of the heme group or reaction with hydrazine reduced but did not eliminate the extraluminal and cytoplasmic reaction product. In contrast, HRP treated with high H2O2 concentrations (0.04 M) was retained in the lumina and cytoplasmic staining was nearly abolished. Immunofluorescent localization of untreated and H2O2-treated HRP after retrograde infusion confirmed the findings obtained using diaminobenzidine procedures. These results suggest that the peroxidatic activity of HRP may damage cell membranes and tight junctions in the rat parotid gland, and indicate that permeability studies employing HRP as a tracer should be interpreted with caution.

Animals↗