Biomedical subjects

S J Hagen

Publications and source records attributed to S J Hagen.

18 recordsLinked to original sources

PubMed1997

Acidic conditions ameliorate both adenosine triphosphate depletion and the development of hyperpermeability in cultured Caco-2BBe enterocytic monolayers subjected to metabolic inhibition.

BACKGROUND: We recently reported that moderate degrees of adenosine triphosphate (ATP) depletion induced by chronic glycolytic inhibition or hypoxia increase the permeability of Caco-2BBe enterocytic monolayers. Interestingly, the development of lactic acidosis induced by anaerobic glycolysis ameliorates the development of hyperpermeability caused by chronic ATP depletion. We sought to further elucidate the mechanism(s) responsible for the apparent protection against epithelial hyperpermeability afforded by mild acidosis under conditions of metabolic inhibition. METHODS: Caco-2BBe monolayers growing on permeable supports in bicameral chambers were incubated with 2-deoxyglucose (2DOG) in a glucose-free (Glu-) environment to inhibit glycolysis. Permeability was determined by measuring the transepithelial flux of fluorescein sulfonic acid. Concentrations of intracellular calcium [Ca2+]i were determined fluorometrically by using fura-2. RESULTS: When extracellular pH (pH0) was maintained at 7.4 or 5.5, incubation of monolayers for 24 hours with Glu-/2DOG increased permeability and depleted intracellular ATP levels. However, keeping pH0 at 7.0 to 6.0 ameliorated both the development of hyperpermeability and the depletion of ATP induced by Glu-/2DOG. These protective effects were observed under acidic conditions created either by addition to the medium of HCl or by incubation under an atmosphere containing 20% CO2. Incubation with Glu-/2DOG caused bulging of the apical membranes of cells (electron microscopy) and derangements in the perijunctional distribution of actin (fluorescence microscopy); however, these structural changes were ameliorated by mild acidosis. Acute chemical hypoxia at pH0 7.4 induced by Glu-/2DOG plus antimycin A decreased cellular ATP levels and elevated [Ca2+]i. Lowering pH0 to 6.8 ameliorated both the depletion of ATP and the increase in [Ca2+]i induced by Glu-/2DOG+antimycin A. CONCLUSIONS: Moderate decreases in pH ameliorate the hyperpermeability induced by metabolic inhibition, possibly by diminishing ATP depletion and blunting increases in [Ca2+]i.

Acidosis↗

PubMed1997

Role of vacuolation in the death of gastric epithelial cells.

The effect of vacuolation on survival of gastric epithelial cells was studied in rabbit gastric glands (RGG) incubated with ammonia and bafilomycin A1, a potent inhibitor of vacuolar ATPase activity. In ammonia, large vacuoles formed and cell survival was reduced to 47.2 +/- 3.4% at 6 h (59.5 +/- 3.8%, buffer). Bafilomycin A1 added at the start to RGG incubated with ammonia inhibited vacuole formation but did not improve cell survival (48.7 +/- 2.8% at 6 h). Bafilomycin A1 added 1-2 h after addition of ammonia reduced the size of vacuoles but did not alter cell survival. Cell survival was not affected by inhibiting protein synthesis. When incubated with ammonia, parietal cells dissociated from the gland and ruptured. After this, chief cells condensed and formed expensive blebs that contained fragmented nuclei. We conclude that 1)ammonia-induced vacuolation of gastric epithelial cells does not influence cell survival, 2) ammonia facilitates necrosis in parietal cells and apoptosis in chief cells, and 3) chief cell survival, in some manner, may be dependent on parietal cells.

Ammonia↗

PubMed1996

Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding.

How fast can a protein fold? The rate of polypeptide collapse to a compact state sets an upper limit to the rate of folding. Collapse may in turn be limited by the rate of intrachain diffusion. To address this question, we have determined the rate at which two regions of an unfolded protein are brought into contact by diffusion. Our nanosecond-resolved spectroscopy shows that under strongly denaturing conditions, regions of unfolded cytochrome separated by approximately 50 residues diffuse together in 35-40 microseconds. This result leads to an estimate of approximately (1 microsecond)-1 as the upper limit for the rate of protein folding.

Cytochrome c Group↗

PubMed1995

Protein reaction kinetics in a room-temperature glass.

Protein reaction kinetics in aqueous solution at room temperature are often simplified by the thermal averaging of conformational substates. These substates exhibit widely varying reaction rates that are usually exposed by trapping in a glass at low temperature. Here, it is shown that the solvent viscosity, rather than the low temperature, is primarily responsible for the trapping. This was demonstrated by placement of myoglobin in a glass at room temperature and subsequent observation of inhomogeneous reaction kinetics. The high solvent viscosity slowed the rate of crossing the energy barriers that separated the substates and also suppressed any change in the average protein conformation after ligand dissociation.

Binding Sites↗

PubMed1995

Comments on the physics and chemistry of trehalose as a storage medium for hemoglobin-based blood substitutes: "from Kramers Theory to the Battlefield".

A glass of the naturally-occurring sugar trehalose may be a suitable medium for the storage of hemoglobin-based blood substitutes. Trehalose has many or possibly all of the properties required for this purpose, including solubilization of hemoglobin to a very high concentration, lack of toxicity, slowing of oxidation to the non-oxygen binding methemoglobin, stability at room temperature and above, and ease of transport. It should also be possible to prepare hemoglobin extremely rapidly for injection into the circulation in situations where blood replacement is required immediately, as in a domestic emergency room or on the battlefield. These practical considerations are briefly discussed, as well as the theoretical reasons for slowing of chemical reactions in the glassy state.

Blood Substitutes↗

PubMed1995

Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane.

Physical-chemical and biological studies of hepatic bile suggest that biliary phospholipid molecules are secreted as unilamellar vesicles. Systematic ultrastructural studies of bile canaliculi were undertaken to visualize this event. Liver tissue was obtained from normal adult male rats (control), from bile salt-depleted rats (by overnight biliary diversion), and from depleted rats infused intravenously with a hydrophilic-hydrophobic congener series of common taurine-conjugated bile salts. Livers were fixed in situ either by modified chemical methods or by ultrarapid cryofixation. In control rats, chemical fixation revealed unilamellar vesicles 63 +/- 17 (+/- SD) nm in diameter, mostly free within canalicular lumena. Vesicles were infrequent in canaliculi of bile salt-depleted rats, but were present in canaliculi of rats infused with taurocholate. In cryofixed liver tissue, vesicles 67 +/- 13 nm in diameter were observed in canaliculi of control rats and bile-salt depleted rats infused with common bile salts. The majority of these vesicles were affixed to the luminal side of the canalicular membrane. The average number of vesicles per bile canaliculus was in agreement with that estimated on the basis of biliary phospholipid secretion rates, mean vesicle size, and area of close-packed phosphatidylcholine molecules. By immunoelectron microscopy, canalicular vesicles were free of actin and of a 100 kDa canalicular membrane protein. We conclude that biliary phospholipid molecules are secreted from hepatocytes into bile canalicular lumena as unilamellar vesicles approximately 63-67 nm in average diameter. We postulate that this secretion mechanism involves lumenal bile salt-induced vesiculation of lipid microdomains in the exoplasmic hemileaflet of the canalicular membrane.

Animals↗

PubMed1994

Handedness: basic physics.

In the News & Comment article "Genetic testing set for takeoff" by Rachel Nowak (22 July, p. 464), Michael Liskay of Oregon Health Sciences University in Portland should have been named as the senior scientist on one of the teams that identified MLH1. The test for Charcot-Marie-Tooth disease detects the duplication of the PMP22 gene, not a deletion, as stated in the table accompanying the article (p. 466).

Magnetics↗

PubMed1994

Localization of brush border cytoskeletal proteins in gastric oxynticopeptic cells from the bullfrog Rana catesbeiana.

The contribution of brush border cytoskeletal proteins (actin, villin, fimbrin, and brush border myosin-1) to organization of the cytoskeletal network underlying apical plications of oxynticopeptic cells was examined by immunohistochemical techniques in frozen sections of gastric mucosa from the bullfrog, Rana catesbeiana. Apical localization of F-actin with phalloidin in oxynticopeptic cells inhibited with cimetidine revealed small, punctate domains within the apical cytoplasm that were consistent with the presence of short microvilli revealed by electron microscopy. Localization of F-actin in cells stimulated with forskolin was limited to a wide continuous band of cytoplasm corresponding to the location of numerous long surface folds. Inhibition of protein synthesis with cycloheximide did not prevent acid secretion or formation of actin filaments within surface folds in stimulated oxynticopeptic cells, suggesting that the formation of filaments does not require actin synthesis. Staining of gastric mucosae with fluorescent DNase-1 demonstrated that oxynticopeptic cells possess an unusually large pool of non-filamentous actin. Taken together, these results suggest that actin-filament formation in stimulated cells occurs by polymerization of an existing pool of non-filamentous actin. Localization of antibodies specific for villin and fimbrin revealed that these proteins were present within intestinal absorptive cells and gastric surface and neck cells but were not present within inhibited or stimulated oxynticopeptic cells. Brush border myosin-1, present in intestinal absorptive cells, was not present in gastric epithelium. Thus, we propose that actin-containing projections in oxynticopeptic cells are not organized like intestinal microvilli and that filament formation occurs after stimulation by modulating intracellular pools of filamentous and non-filamentous actin.

Actins↗

PubMed1994

Exposure of the rat small intestine to raw kidney beans results in reorganization of absorptive cell microvilli.

BACKGROUND/AIMS: A single exposure to raw kidney beans (RKB) results in vesiculation, shortening, and then regrowth of microvilli in the rat small intestine. This study investigated changes that occur in the structure of microvilli 2-10 hours after RKB exposure. METHODS: Circumferences of microvilli from absorptive cells obtained sequentially after challenge with RKB or chow were assigned to one of three groups: small, intermediate, or large. The distribution and concentration of actin in intact mucosae or isolated epithelial sheets were determined by confocal laser scanning microscopy, immunocytochemistry, and immunoblot analysis with specific probes. RESULTS: Six hours after exposure to RKB, most microvilli were large, abnormal in shape, and contained significantly more actin filaments than large microvilli from control rats. In addition, the fluorescence intensity of F-actin increased within injured microvilli without changes in the total intracellular actin concentration. By 8-10 hours after challenge with RKB, some microvilli remained larger than those of control rats but had resumed their normal shape and contained fewer actin filaments than at 6 hours. CONCLUSIONS: Exposure of the rat small intestine to RKB results in enlargement of absorptive cell microvilli and reorganization of membrane and core actin filaments without changes in intracellular actin concentration. Enlarged microvilli are rapidly repaired.

Actins↗

PubMed1991

Class II antigen-associated invariant chain mRNA in mouse small intestine.

MHC class II antigen-associated invariant (Ii) chain mRNA appears in mouse small intestine during postnatal development. Ii chain cDNA hybridizes to RNA from epithelial sheets dissociated from the lamina propria with EDTA. Of several mouse organs tested, only bone marrow and spleen contain higher levels of Ii chain mRNA than small bowel. Ii chain mRNA is not detected in stomach, colon, duodenum, testis, liver, submandibular gland, or L-cell RNA; brain contains a cross-reactive but uncharacterized sequence. cDNA amplification using primers specific for both Ii31 and Ii41 chain mRNAs showed that both forms occur in small intestine. These results support the conclusion that regulation of the class II Ii chain gene is associated with the ontogeny of intestinal immunity.

Animals↗

PubMed1990

Expression of murine CD1 on gastrointestinal epithelium.

Cluster of differentiation 1 (CD1) in humans is a family of major histocompatibility complex (MHC) class I-like molecules expressed on the surface of immature thymocytes, Langerhans cells, and a subpopulation of B cells. The only function identified for human CD1 is as a ligand recognized by a subpopulation of T lymphocytes. In order to study the distribution and function of these molecules in the mouse, a murine CD1 complementary DNA was expressed in mouse fibroblasts and used to produce monoclonal antibodies. These antibodies revealed prominent expression of murine CD1 only on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes. Low levels of expression were also detected on thymocytes and peripheral lymphocytes. The gastrointestinal distribution of murine CD1 suggests that this molecule may be important in epithelial immunity.

Animals↗

PubMed1990

Lowicryl embedding of gastrointestinal tissues.

In the present study, we modified the technique described by Altman et al. (J. Histochem. Cytochem., 32:1217-1223, 1984) for rapid embedding of tissues in Lowicryl K4M. To attain good sections of the small intestine that contained villi, crypts, submucosa, and external muscle layers, we cut 100 micron slices of the full thickness of the wall with a vibratome before embedment and then deoxygenated the resin and tissue before polymerization. The sections we obtained compared favorably with the quality of sections from conventional resins.

Acrylic Resins↗

PubMed1990

Epithelial basement membrane of mouse jejunum. Evidence for laminin turnover along the entire crypt-villus axis.

Little is known regarding turnover of the epithelial basement membrane in adult small intestine. Are components degraded and inserted along the length of the crypt-villus axis or selectively in the crypt region with subsequent migration of basement membrane from crypt to villus tip in concert with epithelium? We injected affinity-purified sheep anti-laminin IgG or sheep anti-laminin IgG complexed to horseradish peroxidase (HRP) into mice to label basement membrane laminin in vivo. Fluorescence microscopy revealed linear fluorescence along the length of the jejunal epithelial basement membrane 1 d after anti-laminin IgG injection. By 1 wk, small nonfluorescent domains were interposed between larger fluorescent domains. Over the next 5 wk the lengths of nonfluorescent domains increased progressively whereas those of fluorescent domains decreased. Additionally, electron microscopy revealed HRP reaction product along the length of the epithelial basement membrane after 1 d whereas unlabeled or lightly labeled domains that increased in length with time were observed interposed between heavily labeled domains by 2 and 4 wk along the entire crypt-villus axis. We conclude that laminin turnover occurs focally in the epithelial basement membrane of mouse jejunum along the crypt-villus axis over a period of weeks and that this basement membrane does not comigrate in concert with its overlying epithelium.

Animals↗

PubMed1989

Repair of microvilli in the rat small intestine after damage with lectins contained in the red kidney bean.

That microvilli of intestinal absorptive cells in the duodenum and jejunum are disrupted by acute challenge with lectins contained in raw kidney beans (RKB) was shown nearly 10 yr ago by light microscopy. However, the precise morphologic damage produced by RKB has not been characterized, and it is not known whether microvilli, once damaged, undergo repair. We have examined these issues by challenging rats with suspensions of 300 mg of RKB, boiled beans, or standard laboratory chow by orogastric lavage. Microvillus length was measured in electron micrographs from 6 to 20 h after challenge. Epithelial cell migration was determined by autoradiography after injection of [3H]thymidine. After challenge with RKB, microvilli (a) showed extensive vesiculation along the length of villi 2-4 h after challenge; (b) were reduced significantly in length along the entire villus 6 h after challenge; and (c) were near normal in length by 20 h after challenge. Microvillus length was also reduced significantly 6 h after challenge with boiled beans. The rate of cell migration was not accelerated by treatment with RKB. These data suggest that damage to microvilli caused by 300 mg of RKB is self-limited and reversible; microvilli once damaged by RKB are repaired. Repair of microvilli is due to intrinsic reparative processes rather than accelerated replacement of damaged cells. We speculate that microvilli may be repeatedly damaged and repaired after ingestion of dietary lectins.

Animals↗

PubMed1988

Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy.

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.

Actins↗

PubMed1987

Demonstration of microtubules in the terminal web of mature absorptive cells from the small intestine of the rat.

The terminal web (TW) region of mature absorptive cells in the small intestine of the rat contains an elaborate cytoskeleton which supports the apical microvillus membrane. In studies regarding the structural organization of the cytoskeleton and associated proteins in the small intestine, microtubules have not been mentioned as components of the TW. By transmission electron microscopy of conventional resin-embedded sections of rat small intestine, we observe many microtubule profiles in the TW of mature absorptive cells. These microtubules are found in various orientations, although most course parallel to the long axis of the cell, and many microtubule profiles are seen in close association with smooth-surfaced vesicles.

Animals↗

PubMed1986

Structural features of absorptive cell and microvillus membrane preparations from rat small intestine.

Absorptive cells of the small intestine are highly polarized cells with distinct microvillus membrane (MVM) and basolateral plasma membrane domains. We compared membrane structure in the following preparations of rat small intestine commonly used for in vitro study of MVM function: epithelial sheets, isolated epithelial cells, and four different MVM vesicle preparations, using electron microscopy of thin sections and freeze fracture replicas. We also quantitated mean vesicle diameter of the four MVM preparations by quasielastic light scattering and determined their actin content. Epithelial sheets maintained their plasma membrane polarity as judged by intramembrane particle (IMP) distribution for at least 30 min after isolation. In contrast, the plasma membrane of isolated cells showed redistribution of IMPs, indicating considerable loss of polarity in the few minutes required for cell recovery. The P-face IMPs in MVM prepared by Ca++ precipitation were randomly distributed but became aggregated after exposure to potassium thiocyanate, which removed approximately 50% of core actin. The P-face IMPs in Mg++ precipitated MVM were aggregated whether or not core actin was depleted with potassium thiocyanate. The shape and size of MVM vesicles differed considerably with different preparative techniques. The extremely rapid loss of plasma membrane polarity of isolated intestinal epithelial cells and the striking structural heterogeneity of MVM vesicles prepared by commonly used techniques should be considered in the interpretation of functional studies with these preparations.

Animals↗

PubMed1986

Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins.

We characterized nine monoclonal antibodies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind to myosin-IB and eight cross-react with Acanthamoeba myosin-II. All but one of the antibodies bind to a 30-kD chymotryptic peptide of myosin-IA that derives from the COOH terminus of the molecule, and to tryptic peptides as small as 17 kD, hence these epitopes are clustered closely together on the heavy chain. None of the antibodies prevent heavy chain phosphorylation by myosin-I heavy chain kinase. One antibody inhibits the K+-EDTA ATPase activity and three antibodies inhibit the actin-activated Mg++-ATPase activity of myosin-I under the set of conditions that we tested. When fluorescent antibody staining of both whole cells and isolated nuclei is done, several of these monoclonal antibodies react strongly with nuclei. These antibodies also stain the cytoplasmic matrix, especially the cortex near the plasma membrane. All nine of the monoclonal antibodies bind to polypeptides of 30-34 kD that are highly enriched in nuclei isolated from Acanthamoeba. There is no myosin-I in the isolated nuclei, so the 30-34-kD polypeptides, not myosin-I, are responsible for the nuclear staining.

Amoeba↗