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G Apodaca

Publications and source records attributed to G Apodaca.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of the Chlamydia trachomatis vacuole and its interaction with the host endocytic pathway in HeLa cells.

Chlamydia trachomatis, an obligate intracellular parasite and a major human pathogen, invades eukaryotic host cells and replicates within a membrane-bound compartment (termed the vacuole or inclusion) in the cytoplasm of the host cell. In this report, we describe in detail the characteristics of the vacuole throughout the chlamydial life cycle in terms of the endocytic pathway, as determined by epifluorescent and confocal immunofluorescence microscopy. By indirect immunofluorescence, the transferrin receptor (TfR), a component of early endosomes, and the cation-independent mannose-6-phosphate receptor (CI-M6PR), a component of late endosomes, were found in close association with the chlamydial vacuole as early as 4 h postinfection (hpi) and as late as 20 hpi. Fluorescein isothiocyanate (FITC)-labeled Tf was also found to colocalize with the vacuole at 4, 12, and 20 hpi, indicating that exogenously added ligands can be transported to the region of the vacuole. Antibodies to several different lysosomal proteins failed to label the chlamydial vacuole at any time point during the life cycle. Indirect immunofluorescence of cells infected with chlamydiae stained with an antibody to the trans-Golgi network (TGN) protein TGN38 demonstrated that in infected cells, the integrity and structure of the TGN was altered. The rates of Tf recycling in infected and uninfected cells were compared by fluorescence microscopy and quantitated with 125I-Tf. While the rate of FITC-Tf recycling from endocytic compartments in chlamydia-infected cells did not appear different from that of uninfected cells, a small pool of FITC-Tf that had accumulated adjacent to the chlamydial vacuole recycled at a slower rate. Quantitation of Tf recycling with 125I-Tf showed that Tf was recycled more slowly in infected cells than in uninfected cells. The altered distribution of several endocytic pathway markers and the slowed Tf recycling are consistent with the hypothesis that the chlamydial vacuole interacts with the endocytic pathway of the host. These results furthermore suggest that the chlamydial vacuole does not correspond to a canonical endocytic compartment but that it is a unique and dynamic organelle that shares several characteristics with recycling endosomes of the host cell. Interactions with the early and/or late endosomal compartments, in addition to the Golgi apparatus, may provide a source of membrane or nutrients for the replicating organisms.

Biomarkers↗
PubMed1996

Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells.

Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N-ethylmaleimide-sensitive factor (NSF)-alpha soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25,000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicle.

Adenosine Triphosphate↗
PubMed1996

Calmodulin regulates the intracellular trafficking in epithelial cells.

In this study we have demonstrated the role of calmodulin in membrane trafficking. By means of specific calmodulin-antagonist (W13, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide) the transcytosis, recycling and receptor-mediated endocytosis of IgA were investigated. In W13 treated MDCK cells, exHpressing the polymeric immunoglobulin receptor (pIgR) and plated on polycarbonate filters (transwells), it has been shown that transcytosis of IgA was inhibited, the recycling was concomitantly increased and there were no modification on its internalization. Altogether, point to a role of calmodulin in regulating the function of endocytic compartment in epithelial cells.

Animals↗
PubMed1995

Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing.

As a model for bacterium-induced epithelial cell injury, we have studied the interaction of Pseudomonas aeruginosa with polarized Madin-Darby canine kidney (MDCK) cells grown on filters. Following an initial period of bacterial adhesion, foci of injured host cells, which consisted of a central region of cell debris, surrounded by cells that were permeable and apparently necrotic, were formed. Host cell death was quantified by measuring the increased permeability of the monolayer to the macromolecular tracer [14C]inulin. Using this MDCK model system, we have identified bacterial and host cell factors necessary for the host cell damage. The ability of P. aeruginosa to cause MDCK cell damage was independent of elastase or exotoxin A production. In contrast, bacteria with a mutation in the regulatory locus exsA (which are deficient in exoenzyme S production) neither bound to nor caused host cell injury. MDCK cells with defects in cell surface glycosylation were resistant to cell injury, indicating that bacteria may require host cell glycolipids and/or glycoproteins as points of adhesion to cause subsequent host cell injury.

Animals↗
PubMed1994

Transcytosis of the polymeric immunoglobulin receptor is regulated in multiple intracellular compartments.

Transcytosis of the polymeric immunoglobulin receptor (pIgR) can be experimentally divided into three steps: 1) internalization from the basolateral plasma membrane and delivery to basolateral early endosomes, 2) microtubule-dependent movement from basolateral early endosomes to apical recycling endosomes, and 3) delivery from apical recycling endosomes to the apical surface and cleavage of the pIgR to secretory component, which is released into the apical medium. Transcytosis of the pIgR is stimulated by two signals, phosphorylation of Ser-664 in the cytoplasmic domain of the pIgR and binding of the ligand, dimeric IgA, to the pIgR. These signals do not detectably alter step 1 of transcytosis. Here, we show that phosphorylation of Ser-664 stimulates both steps 2 and 3, whereas binding of dimeric IgA stimulates only step 3 of transcytosis.

Animals↗
PubMed1994

The calmodulin antagonist, W-13, alters transcytosis, recycling, and the morphology of the endocytic pathway in Madin-Darby canine kidney cells.

The effect of the calmodulin antagonist N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) on the endosomal system of Madin-Darby canine kidney cells was assessed. W-13 inhibited receptor-mediated IgA transcytosis, recycling of transferrin, and caused all material endocytosed from both surfaces of the cell to be delivered to exceptionally large, novel endosomal structures, which appear to be derived from early endosomes. Treatment with other calmodulin antagonists similarly inhibited transcytosis and caused large endosomes to form. These observations raise the possibility that calmodulin may be involved in regulating membrane trafficking through the endosomes of polarized epithelial cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes.

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non-transcytosing molecules.

Animals↗
PubMed1993

Transcytosis of placental alkaline phosphatase-polymeric immunoglobulin receptor fusion proteins is regulated by mutations of Ser664.

Two signals are known that regulate the entry of the polymeric immunoglobulin receptor (pIgR) into the transcytotic pathway: binding of the ligand, dimeric IgA, and phosphorylation of Ser664 in the cytoplasmic domain of the receptor. Mutation of Ser664 to an alanine residue generates a receptor that is transcytosed slowly and exhibits increased recycling at the basolateral plasma membrane. In contrast, if Ser664 is mutated to an aspartate (which bears a constant negative charge-like a phosphate group) the receptor is transcytosed at a rate that is greater than the wild-type pIgR. We have constructed fusions of placental alkaline phosphatase (PLAP) with the transmembrane region and cytoplasmic domain of the wild-type or mutant pIgRs described above (PLAP/WT, PLAP/Ala, and PLAP/Asp) in order to study transcytotic signals present in the cytoplasmic domain of the pIgR independently of those present in the lumenal domain. Transcytosis of the fusion proteins was measured by cell surface immunoprecipitation of a single cohort of basolaterally biotinylated fusion proteins as it arrived at the apical plasma membrane. Significantly more of the PLAP/WT fusion (27%) than the PLAP/Ala fusion protein (5%) was transcytosed during a 2-h chase period. In addition, it was found that approximately 10% of a pseudoligand, monovalent 125I-labeled Fabs (derived from affinity-purified anti-PLAP antibodies), was transcyosed by the PLAP/WT fusion, 7% by the PLAP/Ala fusion, and 22% by the PLAP/Asp fusion over a 4-h period. These data are consistent with the hypothesis that the cytoplasmic domain of the pIgR contains information necessary to direct proteins into the transcytotic pathway, albeit inefficiently, and that phosphorylation of Ser664 is at least a partially autonomous signal for transcytosis.

Alkaline Phosphatase↗
PubMed1993

Brefeldin-A inhibits the delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells.

We have studied the effects of brefeldin A (BFA) on the polarized delivery of the polymeric immunoglobulin receptor to the basolateral surface of MDCK cells. Unlike the delivery of several other basolateral membrane and secretory proteins, the delivery of the polymeric immunoglobulin receptor from the trans-Golgi network to the cell surface was inhibited by BFA. The effect of BFA treatment was apparent at 1.0 microgram/ml (36% inhibition), and maximal inhibition was observed at 10 micrograms/ml (70% inhibition). The delivery of the receptor from the endoplasmic reticulum to the basolateral surface was even more sensitive to the effect of BFA; delivery was inhibited 95% in cells treated with 1 micrograms/ml BFA. The selective action of BFA on the basolateral delivery of the polymeric immunoglobulin receptor suggests that there may be multiple pathways for delivery of proteins to the basolateral cell surface of MDCK cells.

Animals↗
PubMed1993

Apical secretion of a cytosolic protein by Madin-Darby canine kidney cells. Evidence for polarized release of an endogenous lectin by a nonclassical secretory pathway.

In the classical secretory pathway proteins containing a signal peptide are translocated from the cytoplasm of the cell into the lumen of the endoplasmic reticulum (ER). From the ER they are transported to the Golgi apparatus and finally to the plasma membrane (PM) where they are released into the extracellular compartment. However, some proteins are synthesized without a signal peptide and maintain a predominantly cytosolic distribution until they are released from the cell. As a marker for this nonclassical secretory pathway we have chosen L-29, a soluble lectin of M(r) about 29,000, that has affinity for lactose and other beta-galactoside containing glycoconjugates. We were interested in determining if cultured epithelial cells secrete L-29 and if they do so in a polarized fashion. Madin-Darby canine kidney (MDCK)-II cells were found to express large quantities of L-29 (about 1% of the detergent soluble protein). The lectin was diffusely distributed in the cytosol, with little or none in vesicular compartments. The polarity of L-29 secretion, when analyzed in pulse-chase experiments, was selectively into the apical compartment of filter-grown MDCK cells. This secretion was not inhibited by brefeldin A or monensin, drugs that are known to inhibit protein transport through the ER-Golgi-PM pathway. Secretion of L-29 was augmented 3-5-fold by the calcium ionophore A23187 and by increasing the temperature to 42 degrees C, whereas lowering the temperature to 20 degrees C or addition of nocodazole prevented secretion. These results demonstrate the polarized secretion of a cytosolic protein by a nonclassical secretory pathway.

Animals↗
PubMed1992

Secreted inhibitors of metalloproteinases (IMPs) that are distinct from TIMP.

The tissue inhibitor of metalloproteinases (TIMP, M(r) 30,000) is secreted by many cell and tissue types and has been shown to inhibit most secreted mammalian metalloproteinases. In matrix and tissue invasion assays, the inactivation or removal of TIMP enhances invasiveness. However, many of the cells that secrete TIMP also secrete other metalloproteinase inhibitors. By analysis of medium conditioned by various endothelial, mesenchymal, and neural cells on SDS-.substrate-polyacrylamide-inhibitor gels (reverse zymograms), we have detected at least three other distinct inhibitors of metalloproteinases (IMPs). Some or all of these IMPs have been detected in secretions of mouse, rabbit, sheep, and human cells and are all smaller in apparent molecular size than TIMP (IMP-1, M(r) 26,000; IMP-2, M(r) 21,000; IMP-3, M(r) 18,000). These IMPs are not proteolytic degradation products of TIMP nor do they represent nonglycosylated TIMP. The IMPs do not cross-react in the native or denatured state with any of several anti-TIMP antibodies. The IMPs appear to be regulated independently of each other and of TIMP. In vitro, the complex consisting of one of the IMPs, or TIMP, and a metalloproteinase can be dissociated into functional inhibitor and metalloproteinase. Whether this characteristic is significant in vivo is not known. IMP-2 has been purified from several sources and shares sequence homology with TIMP, suggesting that the IMPs and TIMP may constitute a gene family. The most significant characteristic of IMP-2 is that it appears to preferentially inhibit, on a mole:mole basis, the M(r) 68,000 gelatinase rather than collagenase or stromelysin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor.

The polymeric immunoglobulin receptor is normally delivered from the Golgi to the basolateral surface of epithelial cells and then transports polymeric IgA and IgM to the apical surface. We now report that a 14 residue segment of the 103 amino acid cytoplasmic domain, proximal to the plasma membrane, directs the receptor to the basolateral surface. A mutant receptor lacking these 14 amino acids is sorted directly to the apical surface from the Golgi. Furthermore, this sequence is sufficient to redirect an apical membrane protein, placental alkaline phosphatase, to the basolateral plasma membrane. We conclude that this sequence contains an autonomous signal, which specifies sorting from the Golgi to the basolateral surface, a process previously postulated to occur by default.

Alkaline Phosphatase↗
PubMed1990

Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells.

Metalloproteinases have been implicated as important factors mediating the tissue migration of a variety of normal and transformed cells. The conditioned medium (CM) of fetal human astrocytes and five glioma cell lines did not degrade azocoll in suspension, but several proteolytic activities, inhibitable by 1,10-phenanthroline, were detected on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Both cell types secreted three major proteolytic species (Mr 65,000, 57,000, and 52,000). Two of the glioma lines secreted an additional proteinase (Mr 92,000). After treatment with 12-O-tetradecanoylphorbol-13-acetate, the secretion of the Mr 92,000, 57,000, and 52,000 proteinases was induced or enhanced in all of the cells. The Mr 92,000 and 65,000 proteinases bound specifically to a gelatin affinity column. When purified by preparative gel electrophoresis, the Mr 65,000 proteinase was found to degrade type IV procollagen. The Mr 57,000 and 52,000 species were precipitated by anticollagenase IgG. Tissue inhibitor of metalloproteinases was detected in the CM of all of the cells by substrate gel analysis and immunoprecipitation of [35S]methionine-labeled proteins with anti-tissue inhibitor of metalloproteinases IgG. The glioma lines also secreted various amounts of two smaller inhibitors of metalloproteinases (IMPs), also seen in rabbit brain capillary endothelial cell CM (IMP-1 at Mr 22,000 and IMP-2 at Mr 19,000), and an inhibitor not previously identified (IMP-3 at Mr 16,500). 12-O-Tetradecanoylphorbol-13-acetate stimulated the secretion of tissue inhibitor of metalloproteinases in all of the cells and induced IMPs in some of the glioma lines. When gel filtration chromatography of concentrated CM was used to resolve inhibitors from proteinases, the isolated proteinases had activity against azocoll and the glycoprotein and collagen components of an in vitro model of the extracellular matrix. The secretion of a battery of metalloproteinases by astrocytes may be important in facilitating astrocytic migration during development and in pathological conditions such as inflammation or local invasion of astrocytic neoplasms.

Astrocytes↗
PubMed1990

Expression of proteolytic activity by cultures of Trichophyton rubrum.

Trichophyton rubrum parasitizes the keratinized tissues of humans and proteinases secreted by this organism could function to allow this fungus to metabolize and invade host tissues. The effect of culture medium and glucose on the production of proteinases by cultures of T. rubrum was studied and the expression of these enzymes as a function of time in culture was followed. The fungus was grown to stationary-phase in Sabouraud's glucose broth and the original broth was then replaced with neopeptone, elastin salts, or keratin salts medium. The culture medium was assayed for azocollytic, elastinolytic and keratinolytic activity. Proteolytic activity was also analysed by electrophoresis of culture medium on gelatin and elastin substrate gels. T. rubrum expressed relatively high levels of azocoll-, elastin- and keratin-degrading activity in all of the types of media tested. Glucose partially repressed expression of proteolytic activity, the effect being dependent on the time and conditions of culture. Generally, expression of azocollytic activity peaked in the third week of culture, expression of elastinolytic activity peaked in the first or second week of culture, and expression of keratinolytic activity peaked at different times depending on the composition of the medium. These proteinases probably function to provide the fungus with a source of carbon, nitrogen, and sulfur and may also be responsible for the invasion of host tissues by T. rubrum in the rare cases of disseminated disease.

Azo Compounds↗
PubMed1989

Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum.

A proteinase of Mr 27,000 with a possible role in the metabolism and invasion of host tissues was purified from the conditioned medium of Trichophyton rubrum by concanavalin A and anion-exchange chromatography. Peaks of proteolytic activity were analyzed by substrate gel electrophoresis. The 27,000-Mr proteinase had a pH optimum of 8.0, a calcium dependence of 2 mM, and was inhibited by serine proteinase inhibitors, especially phenylmethylsulfonyl fluoride and Phe-Gly-Ala-Leu-chloromethyl ketone. By polyacrylamide gel electrophoresis, the 27,000-Mr proteinase had a reduced molecular weight of 44,000 and reacted with [3H]diisopropyl fluorophosphate. The proteinase degraded azocoll, type III collagen, type IV procollagen, laminin, fibronectin, and the peptide substrates succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (1,573 M-1 s-1) and t-butyloxy carbonyl-Ala-Ala-Leu-p-nitroanilide (1,614 M-1 s-1).

Calcium↗
PubMed1989

Regulation of Trichophyton rubrum proteolytic activity.

Trichophyton rubrum is the most common dermatophyte of humans and normally colonizes the superficial layers of the epidermis (stratum corneum). Several proteinases with a possible role in the metabolism of host proteins have been purified from this fungus. The regulation of these enzymes and their role in fungal metabolism were studied at the biochemical level. General proteolytic (azocollytic) activity was repressed when log-phase cultures of T. rubrum were grown in a minimal medium that contained readily metabolized sources of carbon, nitrogen, sulfur, and phosphorus. When either carbon, nitrogen, or sulfur was deleted from this minimal medium, azocollytic activity was derepressed. In all cases a high-molecular-weight activity (Mr, greater than 200,000) was expressed. A 71,000-Mr proteinase was observed in nitrogen-depleted cultures, and proteolytic species of Mr 124,000 and 27,000 were secreted in sulfur-depleted cultures. The addition of either inorganic (MgSO4, Na2SO3, NaS2O3) or organic (methionine, cysteine) sulfur to the sulfur-depleted medium repressed the expression of azocollytic activity. In contrast, keratinolytic activity was not repressed by carbon, nitrogen, or sulfur but instead was induced when a protein source was included in the minimal medium. Stationary-phase cultures of T. rubrum secreted all proteolytic activities constitutively. Unlike log-phase cultures, the stationary-phase cultures secreted azocollytic, elastinolytic, and keratinolytic activity in minimal medium. These activities fell in the carbon-, nitrogen-, and phosphorous-depleted media but remained high in sulfur-depleted medium. The following model is proposed for the regulation of T. rubrum proteolytic activity. In the initial stages of infection, T. rubrum grows logarithmically. In this state, proteolytic activity is derepressed whenever carbon, nitrogen, or sulfur is lacking in the fungal milieu. The general proteinases produced would act on the nonkeratinous proteins in the stratum corneum. There are probably peptidases, as yet unidentified, that would cleave the peptides generated by the initial proteolysis into amino acids. These amino acids would provide the cell with a source of carbon, nitrogen, and sulfur. Under these conditions, the expression of general proteinases would be repressed, whereas specific keratinases would be induced in this nutrient-rich environment. Disease may occur when the fungus reaches stationary phase, when proteinases are secreted constitutively. These enzymes may directly or indirectly incite a host response, resulting in the inflammatory manifestations of dermatophytosis.

Azo Compounds↗