scholarly journals The subcellular localization of di- and tri-peptide hydrolase activity in guinea-pig small intestine

1970 ◽  
Vol 120 (1) ◽  
pp. 195-203 ◽  
Author(s):  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Brush Border ◽  
Soluble Fraction ◽  
Subcellular Fractionation ◽  
Border Region ◽  
Protein Digestion ◽  
Kinetic Assay ◽  
Brush Borders

1. Two different subcellular fractionation techniques were applied to guinea-pig intestinal mucosa and the composition of the brush borders prepared by the two methods were compared. 2. By using a kinetic assay system the subcellular distribution of activity against ten dipeptides and five tripeptides was studied. 3. Only small amounts (5–10%) of activity against dipeptides were found in the brush-border region, the enzymes being concentrated in the cytosol. 4. Significant amounts (10–60%) of activity against tripeptides were found in the brush border with the remainder largely present in the soluble fraction. 5. The relevance of these studies to the localization in vivo and the possible role of peptidases in protein digestion is discussed.

Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains

1995 ◽  
Vol 108 (12) ◽  
pp. 3775-3786 ◽  
Author(s):  
C. Ruppert ◽  
J. Godel ◽  
R.T. Muller ◽  
R. Kroschewski ◽  
J. Reinhard ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Brush Border ◽  
Subcellular Fractionation ◽  
Actin Binding ◽  
Resting Cells ◽  
Medium Density ◽  
Subcellular Targeting ◽  
Myosin I ◽  
Punctate Pattern

Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin 1. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In subcellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density. The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are sufficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper subcellular localization.

scholarly journals Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders.

1985 ◽  
Vol 100 (5) ◽  
pp. 1647-1655 ◽  
Author(s):  
T C Keller ◽  
K A Conzelman ◽  
R Chasan ◽  
M S Mooseker
Keyword(s):  
Atpase Activity ◽  
Intestinal Epithelium ◽  
Brush Border ◽  
Time Course ◽  
Intestinal Epithelial ◽  
Terminal Web ◽  
Brush Borders

We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37 degrees C under conditions that stimulate terminal web contraction (1 microM Ca++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0 degree C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCl and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only approximately 20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.

scholarly journals Molecular, Biological, and In Vivo Characterization of the Guinea Pig Cytomegalovirus (CMV) Homologs of the Human CMV Matrix Proteins pp71 (UL82) and pp65 (UL83)

2004 ◽  
Vol 78 (18) ◽  
pp. 9872-9889 ◽  
Author(s):  
Alistair McGregor ◽  
Fenyong Liu ◽  
Mark R. Schleiss
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Matrix Proteins ◽  
Wild Type ◽  
Viral Dna ◽  
Guinea Pig Cytomegalovirus ◽  
Human Cmv ◽  
Hsv 1

ABSTRACT We recently identified the genes encoding the guinea pig cytomegalovirus (GPCMV) homologs of the upper and lower matrix proteins of human CMV, pp71 (UL82) and pp65 (UL83), which we designated GP82 and GP83, respectively. Transient-expression studies with a GP82 plasmid demonstrated that the encoded protein targets the nucleus and that the infectivity and plaquing efficiency of cotransfected GPCMV viral DNA was enhanced by GP82. The transactivation function of GP82 was not limited to GPCMV, but was also observed for a heterologous virus, herpes simplex virus type 1 (HSV-1). This was confirmed by its ability to complement the growth of an HSV-1 VP16 transactivation-defective mutant virus in an HSV viral DNA cotransfection assay. Study of a GP82 “knockout” virus (and its attendant rescuant), generated on a GPCMV bacterial artificial chromosome construct, confirmed the essential nature of the gene. Conventional homologous recombination was used to generate a GP83 mutant to examine the role of GP83 in the viral life cycle. Comparison of the one-step growth kinetics of the GP83 mutant (vAM409) and wild-type GPCMV indicated that GP83 protein is not required for viral replication in tissue culture. The role of GP83 in vivo was examined by comparing the pathogenesis of wild-type GPCMV, vAM409, and a control virus, vAM403, in guinea pigs. The vAM409 mutant was significantly attenuated for dissemination in immunocompromised strain 2 guinea pigs, suggesting that the GP83 protein is essential for full pathogenicity in vivo.

scholarly journals Release and fixation of CO2 by guinea-pig kidney tubules metabolizing aspartate

1992 ◽  
Vol 284 (3) ◽  
pp. 697-703
Author(s):  
G Martin ◽  
C Michoudet ◽  
N Vincent ◽  
G Baverel
Keyword(s):  
Guinea Pig ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Co2 Fixation ◽  
Carbon Oxidation ◽  
Kidney Tubules ◽  
Pig Kidney ◽  
Glutamine Synthesis ◽  
Oxoglutarate Dehydrogenase

1. The metabolism of L-[U-14C]aspartate, L-[1-14C]aspartate and L-[4-14C]aspartate was studied in isolated guinea-pig kidney tubules. 2. Oxidation of C-1 plus that of C-4 of aspartate accounted for 90-92% of the CO2 released from aspartate, whereas oxidation of the inner carbon atoms of aspartate (which occurs beyond the 2-oxoglutarate dehydrogenase step) represented only 8-10% of aspartate carbon oxidation. 3. The formation of [1-14C]glutamine and [1-14C]glutamate from [1-14C]aspartate and [4-14C]aspartate indicated that about one-third of the oxaloacetate synthesized from aspartate underwent randomization at the level of fumarate. 4. With [U-14C]aspartate as substrate, the percentage of the C-1 of glutamate and glutamine found radiolabelled after 60 min of incubation was 92.7% and 47.5% in the absence and the presence of bicarbonate respectively. 5. That CO2 fixation occurred at high rates in the presence of bicarbonate was demonstrated by incubating tubules with aspartate plus [14C]bicarbonate; under this condition, the label fixed was found in C-1 of glutamate, glutamine and aspartate, as well as in C-4 of aspartate, demonstrating not only randomization of aspartate carbon but also aspartate resynthesis secondary to oxaloacetate cycling via phosphoenolpyruvate carboxykinase, pyruvate kinase and pyruvate carboxylase. 6. The importance of CO2 fixation in glutamine synthesis from aspartate is discussed in relation to the possible role of the guinea-pig kidney in systemic acid-base regulation in vivo.

Intestinal absorption of ester and ether glycerophospholipids in guinea pig. Role of a phospholipase A2 from brush border membrane

Lipids ◽  
1987 ◽  
Vol 22 (1) ◽  
pp. 33-40 ◽  
Author(s):  
A. Diagne ◽  
S. Mitjavila ◽  
J. Fauvel ◽  
H. Chap ◽  
L. Douste-Blazy
Keyword(s):  
Guinea Pig ◽  
Phospholipase A2 ◽  
Intestinal Absorption ◽  
Brush Border ◽  
Brush Border Membrane

L-649,923, Sodium (βS*, γR*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propylthio)-γ-hydroxy-β-methylbenzenebutanoate, a selective, orally active leukotriene receptor antagonist

1986 ◽  
Vol 64 (8) ◽  
pp. 1068-1075 ◽  
Author(s):  
T. R. Jones ◽  
R. Young ◽  
E. Champion ◽  
L. Charette ◽  
D. Denis ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Plot Analysis ◽  
Prostaglandin F ◽  
Leukotriene Receptor ◽  
Orally Active ◽  
Intraduodenal Administration

L-649,923, Sodium (βs*, γR*)-4-(3-(4-acetyi-3-hydroxy-2-propylphenoxy)propylthio)-γ-hydroxy-β-methylbenzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (Ki value of 400 nM) and to a lesser extent [3H]leukotriene C4 (Ki value of 8.6 μM) binding in guinea-pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotriene C4, D4, E4, and F4 but not those induced by acetylcholine, histamine, serotonin, prostaglandin F2α, or U-44069 (stable endoperoxide analogue). Schild plot analysis indicated a competitive inhibition of contractions of guinea-pig ileum induced by leukotriene D4 (pA2 8.1) and contractions of guinea-pig trachea induced by leukotrienes E4 and F4 (pA2 7.1 and 6.9, respectively). In contrast, contractions of guinea-pig trachea induced by leukotrienes C4 (pA2 7.2; slope 0.6) and D4 (pA2 7.2; slope 0.7) were inhibited in a noncompetitive fashion. In vivo, intravenously administered L-649,923 selectively blocked bronchoconstriction induced in anesthetized guinea pigs by leukotriene C4 and D4 (ED50 values i.v. 0.38 and 0.26 mg/kg, respectively) but not that induced by histamine, arachidonic acid, serotonin, U-44069, or acetylcholine. Following intraduodenal administration, L-649,923, blocked leukotriene D4 induced bronchoconstriction (5 and 10 mg/kg). The present findings indicate that selective antagonists, such as L-649,923, may be useful for defining the role of leukotrienes in diseases such as bronchial asthma.

scholarly journals Galectin-4 and Small Intestinal Brush Border Enzymes Form Clusters

1997 ◽  
Vol 8 (11) ◽  
pp. 2241-2251 ◽  
Author(s):  
E. Michael Danielsen ◽  
Bo van Deurs
Keyword(s):  
Brush Border ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Intestinal Lumen ◽  
Immunogold Electron Microscopy ◽  
Brush Border Enzymes ◽  
Intestinal Brush Border ◽  
Mucosal Explants

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

Membrane repolarization is delayed in proximal tubules after ischemia-reperfusion: possible role of microtubule-organizing centers

2003 ◽  
Vol 285 (2) ◽  
pp. F230-F240 ◽  
Author(s):  
Flavia A. Wald ◽  
Yolanda Figueroa ◽  
Andrea S. Oriolo ◽  
Pedro J. I. Salas
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Ischemia Reperfusion ◽  
Proximal Tubules ◽  
Microtubule Organizing Centers ◽  
Apical Domain ◽  
Brush Borders ◽  
Chemical Anoxia

We have previously shown that microtubule-organizing centers (MTOCs) attach to the apical network of intermediate filaments (IFs) in epithelial cells in culture and in epithelia in vivo. Because that attachment is important for the architecture of microtubules (MTs) in epithelia, we analyzed whether chemical anoxia in LLC-PK1 and CACO-2 cells or unilateral ischemia-reperfusion in rat kidney (performed under fluorane anesthesia) had an effect on the binding and distribution of MTOCs. In culture, we found that chemical anoxia induces MTOC detachment from IFs by morphological and biochemical criteria. In reperfused rat proximal tubules, noncentrosomal MTOCs were fully detached from the cytoskeleton and scattered throughout the cytoplasm at 3 days after reperfusion, when brush borders were mostly reassembled. At that time, MTs were also fully reassembled but, as expected, lacked their normal apicobasal orientation. Two apical membrane markers expressed in S2 and S3 segments were depolarized at the same stage. At 8 days after reperfusion, membrane polarity, MTOCs, and MTs were back to normal. Na+-K+-ATPase was also found redistributed, not to the apical domain but rather to an intracellular compartment, as described by others (Alejandro VS, Nelson W, Huie P, Sibley RK, Dafoe D, Kuo P, Scandling JD Jr., and Myers BD. Kidney Int 48: 1308–1315, 1995). The prolonged depolarization of the apical membrane may have implications in the pathophysiology of acute renal failure.

scholarly journals Adenosine-mediated hypotension in in vivo guinea-pig: receptors involved and role of NO

2001 ◽  
Vol 134 (4) ◽  
pp. 745-752 ◽  
Author(s):  
P Nieri ◽  
E Martinotti ◽  
V Calderone ◽  
M C Breschi
Keyword(s):  
Guinea Pig
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