Dynamic Expression of Genes Involved in Proteoglycan/Glycosaminoglycan Metabolism during Skin Development.

Glycosaminoglycans are important for cell signaling and therefore for proper embryonic development and adult homeostasis. Expressions of genes involved in proteoglycan/glycosaminoglycan (GAG) metabolism and of genes coding for growth factors known to bind GAGs were analyzed during skin development by microarray analysis and real time quantitative PCR. GAG related genes were organized in six categories based on their role in GAG homeostasis, viz. (1) production of precursor molecules, (2) production of core proteins, (3) synthesis of the linkage region, (4) polymerization, (5) modification, and (6) degradation of the GAG chain. In all categories highly dynamic up- and downregulations were observed during skin development, including differential expression of GAG modifying isoenzymes, core proteins, and growth factors. In two mice models, one overexpressing heparanase and one lacking C5 epimerase, differential expression of only few genes was observed. Data show that during skin development a highly dynamic and complex expression of GAG-associated genes occurs. This likely reflects quantitative and qualitative changes in GAGs/proteoglycans, including structural fine tuning, which may be correlated with growth factor handling.

Cytochalasin-induced actin disruption of polarized enterocytes can augment internalization of bacteria.

Cytochalasin-induced actin disruption has often been associated with decreased bacterial internalization by cultured epithelial cells, although polarized enterocytes have not been systematically studied. In assays using confluent polarized HT-29 enterocytes, cytochalasin D appeared to increase internalization of wild-type Salmonella typhimurium, Proteus mirabilis, and Escherichia coli. HeLa and HEp-2 epithelial cells, as well as HT-29 and Caco-2 enterocytes, were used to clarify this unexpected observation. Resulting data showed that cytochalasin D was associated with increased internalization of S. typhimurium and P. mirabilis by both HT-29 and Caco-2 enterocytes and with increased internalization of E. coli by HT-29 enterocytes; with either HeLa or HEp-2 cells, cytochalasin was associated with no change or a decrease in internalization of these same bacterial strains. Cytochalasin caused decreased internalization of Listeria monocytogenes by HT-29, Caco-2, HeLa, and HEp-2 cells, indicating that cytochalasin did not consistently augment bacterial internalization by polarized enterocytes. Fluorescein-labeled phalloidin confirmed marked disruption of filamentous actin in cytochalasin-treated HT-29, Caco-2, HeLa, and HEp-2 cells. Cytochalasin had no noticeable effect on epithelial viability but caused distorted apical microvilli, cell rounding, and separation of adjacent enterocytes in confluent cultures (with a corresponding decrease in transepithelial electrical resistance). Scanning electron microscopy showed that cytochalasin-induced enhanced bacterial internalization was associated with preferential bacterial adherence on the exposed enterocyte lateral surface. Colchicine, used to disrupt microtubules, had no noticeable effect on bacterial internalization by HT-29 or Caco-2 enterocytes. These data indicated that for HT-29 and Caco-2 enterocytes, cytochalasin-induced disruption of filamentous actin might augment internalization of some bacterial species by a mechanism that appeared to involve exposure of the enterocyte lateral surface.

Intracellular survival of enteric bacteria in cultured human enterocytes.

Translocating enteric bacteria have been visualized within intact intestinal epithelial cells in animal models of bacterial translocation. Although the ability of the enterocyte to engulf bacteria has been well documented in both in vivo and in vitro experimental models, relatively little is known about the enterocyte's ability to degrade internalized bacteria. Intracellular survival of eight strains of enteric bacteria (two strains of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, two strains of Escherichia coli, and two strains of Enterococcus faecalis) was quantified over a 20 h period using two different types of terminally differentiated polarized enterocytes considered relevant in vitro models of human small intestinal epithelium, namely Caco-2 and HT-29 cells. Caco-2 enterocytes were generally more permissive for bacterial uptake when compared with HT-29 enterocytes. However, bacterial survival was similar within each type of enterocyte, and most strains of enteric bacteria remained viable within enterocytes for the 20 h duration of the assay. In addition, with the exception of one strain of L. monocytogenes in Caco-2 cells, intracellular enteric bacteria had no noticeable effect on host enterocyte viability for this 20 h duration. Transmission electron microscopy was used to visualize both intact and degraded bacteria within individual enterocytes, suggesting that prolonged bacterial survival might have resulted from simultaneous bacterial degradation and replication. Thus, although enterocytes internalize enteric bacteria, enterocytes might not kill internalized bacteria as efficiently as leukocytes. Observations of bacterial intracellular survival supported the hypothesis that the enterocyte might be a portal of entry for translocating microbes, and observations of intracellular bacterial degradation might have implications for the role of the enterocyte as an antigen-presenting cell.

Bacteroides fragilis enterotoxin modulates epithelial permeability and bacterial internalization by HT-29 enterocytes.

BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis has been associated with diarrheal disease, and the enterotoxin has a cytopathic effect on cultured HT-29 enterocytes. Experiments were designed to determine the effect of B. fragilis enterotoxin on bacteria-enterocyte interactions. METHODS: Confluent HT-29 enterocytes were incubated for 1 hour with B. fragilis enterotoxin, followed by 1 hour of incubation with pure cultures of enteric bacteria, namely, Salmonella typhimurium (two strains), Listeria monocytogenes (three strains), Proteus mirabilis, Escherichia coli (three strains), and Enterococcus faecalis. Enterocyte viability was assessed using vital dyes, epithelial permeability was measured using transepithelial electrical resistance, enterocyte morphology and bacteria-enterocyte interactions were visualized using light and electron microscopy, and bacterial internalization was assessed using a quantitative culture of lysed enterocytes. RESULTS: B. fragilis enterotoxin did not affect enterocyte viability but decreased transepithelial electrical resistance, and individual enterocytes pulled apart. Enterotoxin pretreatment decreased internalization of L. monocytogenes (P < 0.01) but increased (P < 0.01) internalization of the other strains of enteric bacteria. Augmented bacterial internalization was associated with preferential bacterial adherence on the exposed lateral surface of enterotoxin-treated enterocytes. CONCLUSIONS: B. fragilis enterotoxin was associated with HT-29 cell rounding and with augmented internalization of selected strains of enteric bacteria that were preferentially adherent on the exposed enterocyte lateral surface.

Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells.

Cholecystokinin (CCK) receptors on rat pancreatic acinar cells display two binding affinity states in the presence of adeninine and guanine triphosphates with the effect of ATP mediated by the enzyme nucleoside diphosphate kinase. To determine whether this behavior was intrinsic to a single receptor protein we studied the binding affinity of CHO cells stably transfected with a cloned rat CCKA receptor. 125I-CCK binding to intact cells at 37 degrees C revealed two affinity states for CCK of Kd values 20 pM and 2.4 nM. Membranes prepared from these cells displayed a single affinity state for CCK but two affinity states could be restored in the presence of GTP[gamma S], ATP and ATP[gamma S] but not AMP-PCP. ATP and ATP[gamma S] but not AMP-PCP were substrates for nucleoside diphosphate kinase present in CHO cell membranes and transferred their terminal phosphate to GDP. These findings indicate that the interconvertible affinity states of the CCK receptor are inherent in a single receptor protein and that nucleoside diphosphate kinase mediates the effect of ATP to regulate these two affinity states.

Exposure of the lateral enterocyte membrane by dissociation of calcium-dependent junctional complex augments endocytosis of enteric bacteria.

Intestinal bacterial translocation is facilitated in a variety of clinical conditions involving increased intestinal permeability, such as shock and trauma. Because there is both in vivo and in vitro evidence that enteric bacteria can be internalized by intestinal epithelial cells, experiments were designed to test the effect of increased intestinal permeability on enterocyte endocytosis of enteric bacteria. Mature, confluent cultures of HT-29 enterocytes were placed in a calcium-free solution for 1 h. Enterocyte viability was not noticeably altered, but transepithelial electrical resistance was significantly decreased (indicating a decrease in epithelial junctional integrity), and the enterocytes were pulled apart. Electron microscopic observations revealed enteric bacteria preferentially adherent on the exposed enterocyte lateral surface, and the numbers of viable enteric bacteria (Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, Escherichia coli, and Enterococcus faecalis) internalized by these enterocytes were significantly increased. Restoration of calcium restored confluency to enterocyte cultures, and bacterial internalization reverted to control levels. Thus, calcium-dependent junctional integrity might play a role in augmenting bacterial translocation in clinical conditions associated with increased intestinal permeability.

Nucleoside diphosphate kinase associated with rat pancreatic membranes regulates CCK receptor affinity.

We have previously demonstrated in permeabilized rat pancreatic acini that the existence of two affinity states of the pancreatic cholecystokinin (CCK) receptor seen in intact cells depends on the presence of ATP. In the present study, we demonstrate that this effect of ATP is mediated by the enzyme nucleoside diphosphate kinase (NDPK). Northern blot hybridization analysis demonstrated NDPK mRNA in pancreas. Furthermore, pancreatic membranes possessed NDPK activity, which transferred high-energy phosphate groups to [8-3H]GDP. This enzyme also utilized UTP and ITP as a source of gamma-phosphate for GTP formation while guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was formed in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). However, adenylyl (beta, gamma-methylene)-diphosphate (AMP-PCP) did not serve as a substrate for NDPK. Analysis of 125I-Bolton-Hunter-labeled CCK octapeptide ([125I]BH-CCK-8) binding data in the absence of nucleotides was consistent with a single affinity state with dissociation constant (Kd) equal to 80 pM and maximal binding equal to 50.8 fmol/mg. ATP, UTP, ITP, ATP gamma S, and GTP gamma S all induced two CCK binding affinity states, which in the presence of 1 mM ATP were Kd = 74 pM for high-affinity sites and Kd = 4.3 nM for low-affinity sites: AMP-PCP did not induce two affinity states. GDP at 10 microM had no effect on CCK binding but potentiated the effect of ATP. GTP gamma S, in addition to inducing high- and low-affinity states, also elicited a significant concentration-dependent reduction in the total number of measurable CCK receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cholecystokininA receptor agonist activity by a family of cholecystokininB receptor antagonists.

The dipeptoid cholecystokinin (CCK)B antagonists PD136450, Cam-1279 and CI988 stimulated amylase release from isolated rat pancreatic acini with an efficacy similar to CCK8, but with a much weaker potency (ED50, 0.6, 0.9 and 1.3 microM, respectively). In contrast to CCK8, however, none of these compounds elicited inhibition of amylase release at supramaximal concentrations. In addition, 10(-4) M PD136450 blocked the inhibition induced by high concentrations of CCK8. Competitive inhibition of [125I]BH-CCK8 binding by PD136450 indicated that this compound bound with a single affinity state to all CCK receptors on acini. Maximal stimulation of amylase release by PD136450 was dependent upon occupation of virtually the entire complement of CCK receptors. PD136450 at all concentrations examined had only a limited ability to stimulate total phosphoinositide hydrolysis and at maximum induced only 20% of maximal CCK stimulation. Measurement of intracellular calcium ([Ca++]i) by digital imaging of Fura-2 indicated that 1 microM PD136450 induced repetitive [Ca++]i oscillations with a magnitude of 346.0 +/- 4.5 nM and frequency of 1.3 cycles per min. These oscillations were still present in Ca(++)-free medium and were blocked by the phospholipase C inhibitor, U73122. Because the dipeptoid compounds can occupy all available pancreatic CCKA receptors, these compounds must induce a configuration of the receptor different from either CCK8 or the previously characterized partial agonist CCK-JMV-180, thereby inducing a distinct signaling pattern. Because the dipeptoid compounds do not fully mimic CCK actions, it is likely that they interact with only some of the critical binding sites within the CCKA receptor normally occupied by CCK8.

Molecular identification of the liver- and the heart-type fatty acid-binding proteins in human and rat kidney. Use of the reverse transcriptase polymerase chain reaction.

The cDNAs of two types of fatty acid-binding protein (FABP) present in human kidney, previously described as types A and B, were isolated using reverse transcriptase-PCR (RT-PCR) with human kidney mRNA and various sets of primers. The cDNA fragments were cloned and sequenced. Renal FABP type A and B cDNAs appeared to be completely identical to human liver- and heart-type FABP cDNAs respectively. In the second part of this study we demonstrated the presence of liver-type FABP in rat kidney by chromatography, e.l.i.s.a. and immunocytochemistry. The ratio and cellular distribution of the two FABP types varies markedly in human and rat kidney. Using RT-PCR we were also able to prepare and identify liver- and heart-type FABP cDNAs with mRNA from both male and female rat kidney.