Growth retardation, duodenal lesions, and aberrant ileum architecture in triple null mice lacking EGF, amphiregulin, and TGF-alpha.

BACKGROUND AND AIMS: Mice lacking epidermal growth factor (EGF), transforming growth factor alpha, and amphiregulin were used to identify roles for these EGF receptor (EGF-R) ligands in gastrointestinal development and mucosal integrity. METHODS: Gastrointestinal tract development was examined in knockout mice and correlated with expression of EGF-R protein and EGF family members throughout the gut. Crossfostering experiments addressed roles of maternal- and neonatal-derived ligands in pup growth and intestinal development. Cysteamine-induced ulceration in EGF(-/-) mice was used to examine its role in mucosal cytoprotection. RESULTS: Neonatal mice lacking all 3 ligands were growth retarded, even when reared by wild-type dams; conversely, lack of maternal ligands transiently impaired wild-type pup growth. Triple null neonates displayed spontaneous duodenal lesions, and ileal villi were truncated and fragile with reduced cellular proliferation in the crypts. However, maturation of digestive enzymes was unaffected. Adult EGF(-/-) mice displayed more severe lesions in response to cysteamine treatment compared with wild-type counterparts, although triple null mice were not more susceptible to dextran sulfate sodium-induced colitis, suggesting a differential role for these ligands in the injury response. CONCLUSIONS: EGF-R ligands are required for development and mucosal maintenance in mouse small intestine. Both maternal and neonatal sources of growth factors are required for optimal pup growth.

Denileukin diftitox.

Primary cutaneous T-cell lymphomas (CTCLs) encompass a wide variety of lymphomas that are characterized by the localization of the malignant lymphocytes to the skin at presentation. They are slow-growing and rare, occurring in fewer than 1,000 people annually. Patients may go for months to years with skin abnormalities before being diagnosed. Mycosis fungoides and Sezary syndrome are the most common forms of CTCL and are considered to be indolent diseases. Patients with T1 disease have a normal life expectancy, whereas patients who undergo transformation to large cell lymphoma (8%-23% of patients) have a poor prognosis, with mean survival ranging from 2-19 months.

Mutagenesis reveals a role for epidermal growth factor receptor extracellular subdomain IV in ligand binding.

The extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) comprises four subdomains (I-IV) and mediates binding of several different polypeptide ligands, including EGF, transforming growth factor-alpha, and heparin-binding EGF. Previous studies have predominantly implicated subdomain III in ligand binding. To investigate a possible role for sequences in subdomain IV, we constructed several mutant EGFRs in which clusters of charged or aromatic amino acids were replaced with alanine. Analysis of stably transfected Chinese hamster ovary cells expressing mutant EGFRs confirmed that they were present on the cell surface at levels approaching that of the wild-type receptor. Although tyrosine phosphorylation of most mutants was markedly induced by EGF, a cluster mutation (mt25) containing four alanine substitutions in the span of residues 521-527 failed to respond. EGF-induced tyrosine phosphorylation of an alternative mutant (DeltaEN) with amino acids 518-589 deleted was also greatly diminished. Larger doses of EGF or heparin-binding EGF induced only weak tyrosine phosphorylation of mt25, whereas the response to transforming growth factor-alpha was undetectable. These results suggest that mt25 might be defective with respect to either ligand binding or receptor dimerization. Quantitative analyses showed that binding of (125)I-EGF to mt25 and DeltaEN was reduced to near background levels, whereas binding of EGF to other cluster mutants was reduced 60-70% compared with wild-type levels. Among the mutants, only mt25 and DeltaEN failed to form homodimers or to transphosphorylate HER2/Neu in response to EGF treatment. Collectively, our results are the first to provide direct evidence that discrete subdomain IV residues are required for normal binding of EGF family ligands. Significantly, they were obtained with the full-length receptor in vivo, rather than a soluble truncated receptor, which has been frequently used for structure/function studies of the EGFR extracellular region.

The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system.

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.

Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells.

The molecular mechanisms underlying protein kinase C (PKC) isozyme-mediated control of cell growth and cell cycle progression are poorly understood. Our previous analysis of PKC isozyme regulation in the intestinal epithelium in situ revealed that multiple members of the PKC family undergo changes in expression and subcellular distribution precisely as the cells cease proliferating in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC isozyme(s) in control of intestinal epithelial cell growth and cell cycle progression was examined directly using the IEC-18 immature crypt cell line as a model system. Treatment of IEC-18 cells with PKC agonists resulted in translocation of PKC alpha, delta, and epsilon from the soluble to the particulate subcellular fraction, cell cycle arrest in G1 phase, and delayed transit through S and/or G2/M phases. PKC-mediated cell cycle arrest in G1 was accompanied by accumulation of the hypophosphorylated, growth-suppressive form of the retinoblastoma protein and induction of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC alpha, delta, and epsilon. Differential down-regulation of individual PKC isozymes revealed that PKC alpha in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC alpha in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ.

The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme-specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.

Adaptations of myocardial beta-adrenergic receptor complex in hibernating marmots.

Properties of marmot (Marmota flaviventris) myocardial beta-adrenergic receptor complex (beta-AR) were evaluated during hibernation (H), in summer (S) animals, and in animals aroused from hibernation (C). The results obtained for S and C animals were identical, and only the results for C animals are shown. In H-animal myocardial membrane preparations assayed at 37 degrees C, isoproterenol-dependent adenylate cyclase activity (ACA) was consistently higher, whereas the synergistic contribution of 5'-guanylylimidodiphosphate [Gpp(NH)p] in this reaction was reduced. When assayed at 10 degrees C, only the ACA in H animals responded to the combination of isoproterenol and Gpp(NH)p. In contrast, at 10 degrees C, ACA in response to Gpp(NH)p alone is essentially equal in H and C animals. Hibernation did not change myocardial beta-AR receptor density or affinity. In contrast, analysis of isoproterenol displacement of [125I]iodocyanopindolol revealed that the proportion of beta-AR in the high-affinity state was substantially greater in H than in C animals, and this relationship was retained even in the presence of Gpp-(NH)p. In an evaluation of the role of the GTP binding proteins that couple the beta-AR to the effector adenyl cyclase, we determined that there was no change in the cholera toxin- or pertussis toxin-dependent ADP ribosylation patterns. Immunochemical detection of the individual GTP binding proteins revealed no change in the levels of G alpha i1, G alpha i2, or G alpha i3. In contrast, we observed a hibernation-associated decrease in G alpha o associated with the plasma membrane-enriched particulate fraction. (ABSTRACT TRUNCATED AT 250 WORDS)

Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy.

Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.

Stimulation of calcium influx in HL60 cells by cholesteryl-modified homopolymer oligodeoxynucleotides.

Cholesteryl-modified 15-mer homopolymers of cytidine and thymidine phosphodiester oligodeoxynucleotides (chol-OdC15 and chol-OdT15), but not chol-modified heteropolymeric oligos or chol-modified phosphorothioate oligos, were found to increase cytosolic free Ca2+ in HL60 cells. A flow cytometer and the calcium-sensitive dye indo-1 were used to make multiparameter measurements on the HL60 cells. Chol-OdC15 (5-10 microM) triggered a rapid increase (within 1 min) in [Ca2+]i, with a subsequent slow decline to baseline over 15 min in the continuous presence of agonist. The effect was preserved after unloading the intracellular Ca2+ stores with caffeine and ryanodine. The effect was not sensitive to membrane depolarization by KCl (60 mM) or nimodipine, a dihydropyridine calcium channel antagonist. An increase in [Ca2+]i was absent in a Ca(2+)-free solution and was inhibited by the inorganic Ca2+ channel blocker Cd2+. The results suggest that Ca2+ influx activated by the chol-oligomer is probably mediated by receptor-operated Ca2+ channels. This effect may be due to direct binding of the chol-oligo to the channel or to induced conformational changes due to modification of the local microenvironment.

Two subtypes of dihydropyridine-sensitive calcium channels in rat ventricular muscle.

Two opposite inotropic effects of the dihydropyridine activators, CGP 28392 and Bay K 8644, given at the same concentration (1-2 microM) were found in rat papillary muscles: a positive effect in polarized tissue (4 mM KCl) and a negative one during partial depolarization. The depressive effect found at a low rate or after a short rest was associated with marked prolongation of the Ca2(+)-mediated action potential, indicating that the drugs behave as Ca channel stimulators. The depressive effect of the activation on the resting state contraction was antagonized by nifedipine (2 microM) and high Mg2+ (5 mM). It was suggested that at least two subtypes of the L-type, dihydropyridine-sensitive channels underlie the opposite inotropic responses of the activators. The positive effect is apparently caused by conventional stimulation of Ca2+ entry through the cell membrane, whereas the negative effect is probably due to the stimulation of Ca2+ efflux from the sarcoplasmic reticulum, leading to depletion of intracellular stores. The effect was proposed to be mediated by activation of junctional channels linked to sarcoplasmic reticulum Ca2+ release. An important role for these channels in triggering the sarcoplasmic reticulum Ca2+ release and regulation of force-frequency relation is proposed.

Positive inotropic effect of ryanodine on rabbit ventricular muscle: dependence on the intracellular calcium load.

Two types of electrical and mechanical responses to 1 mumol/l ryanodine, depending on the intracellular calcium load, were observed in rabbit papillary muscles. In a normal calcium solution, ryanodine induced a transient decline followed by a stable increase in the developed force (by 20 +/- 5% of the pretreatment level; n = 30) and prolonged the action potential (AP). The positive ryanodine response showed an increased time-to-peak force and was completely suppressed by 2 mumol/l nifedipine, partially blocked by 50 mumol/l tetracaine (Ca2+ release blocker), but greatly potentiated by 20 mmol/l CsCl or (-) Bay R 5414 which prolonged the AP. The prolonged time-to-peak force of the positive ryanodine response was shortened by procedures raising the content of Ca2+ in the sarcoplasmic reticulum (SR). It is suggested that the initial decline in the force amplitude results from Ca2+ leakage from the SR which is further compensated for by an elevation of both the transmembrane Ca2+ entry and intracellular Ca2+ release. In calcium overloaded myocardium, 1 mumol/l ryanodine caused irreversible contracture and dramatic AP shortening, explained by a massive Ca2+ release from the overloaded SR into the cytoplasm. It is concluded that the calcium content in the SR is the main modulator of the electrical and mechanical effects of ryanodine in ventricular myocardium.

Ca2+-antagonistic properties of phospholipase A2 inhibitors, mepacrine and chloroquine.

The effects of putative phospholipase A2 inhibitors mepacrine and chloroquine on membrane ionic currents were studied in intact frog atrial trabeculae. Both agents decreased slow calcium channel current Isi and fast sodium channel current If. Isi was affected twice at least in comparison to If. Half-block of Isi was observed at approximately 10(-6) mol/l mepacrine and at approximately 10(-5) mol/l chloroquine. These effects on transmembrane ionic transport should be considered when using the above agents as phospholipase inhibitors or antiarrhythmic drugs.

Paradoxical reversion of the inhibitory effects of dihydropyridine enantiomers on the calcium current in frog heart by CGP 28861.

A dihydropyridine CGP 28861 (5 x 10(-6) M) did not change slow inward Ca current as measured by the double sucrose gap method in frog atrial fibres but decreased the agonist effects of Bay K 8644, CGP 28392, (+)-(S)-202-791 and the antagonist effects of nifedipine and (+)-(PN)-200-110. Paradoxically, the weak antagonists (-)-(R)-202-791, (-)-(PN)-200-110 and (+)-Bay K 8644 increased Ca-current after washout of CGP 28861. These data suggest that CGP 28861 can convert the dihydropyridine Ca-channel receptor from an antagonistic site into an agonistic one.

Ryanodine as a trigger of tension oscillations in rat ventricular muscle.

In rat ventricular muscles, ryanodine (10-30 nM) evoked tension oscillation in the relaxation phase of an isometric twitch (relaxation oscillations) and an increase of tonic tension (ryanodine contracture). Both events were more pronounced in Ca2+-loaded rat muscles due to the addition of 0.5 microM adrenaline, an increase in the Ca2+ concentration in the solution and high muscle activity. The ryanodine-induced tension oscillations were comparable to those triggered by caffeine in this species. In both cases, blockers of the release of Ca2+ from the sarcoplasmic reticulum, namely tetracaine and dantrolene, abolished the relaxation oscillations and the ryanodine contracture. The results suggest that the ability of low concentrations of ryanodine to facilitate the release of Ca2+ from the sarcoplasmic reticulum, as shown recently in biochemical experiments, makes a direct contribution to triggering the relaxation oscillations and the ryanodine contracture in intact ventricular muscles. The Ca2+ load of the sarcoplasmic reticulum appears to be essential for the manifestation of the ryanodine Ca2+-releasing activity in rat ventricular muscles.

Ryanodine in low concentrations is a Ca-release stimulator rather than inhibitor in rat myocardium.

The effects of ryanodine on negative force staircase and potentiated rested-state contraction (RC) in rat myocardium were compared to the action of Ca release stimulator (caffeine) and inhibitors (local anesthetics). Only low ryanodine concentrations (0.1-0.5 mumol/l) were found to reverse anomalous mechanical patterns in rat myocardium to similar to those as generally observed in other mammalian species. Ryanodine-induced positive staircase and a weak RC were potentiated by noradrenaline. The results obtained seem to characterize ryanodine as a Ca2+ release stimulator rather than an inhibitor in this species and suggest different molecular substrates for ryanodine and caffeine inotropy in rat myocardium.

Contribution of a Ca-dependent component to the transient outward current in rabbit ventricular fibres.

To evaluate a Ca-sensitive component of the transient outward current, we studied action potentials, isometric tension, and membrane currents (single sucrose gap voltage clamp) of rabbit ventricular heart muscle in the presence of Ryanodine that selectively blocks the intracellular Ca release. Ryanodine (1 microM) prolonged the post rest (10 s) action potential, abolished the "notch" preceding the plateau phase, and depressed the isometric tension. With the same protocol a diminished transient outward current was measured. Application of 1 mM 4-aminopyridine in the presence of Ryanodine further prolonged the duration of the action potential, decreased the transient outward current but increased the maximal tension. It is concluded that in rabbit ventricular myocardium both a Ca-dependent component and a 4-aminopyridine sensitive component compose the transient outward current; however, the 4-aminopyridine sensitive component is dominating.