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PubMed1984

Regulation through phosphorylation/dephosphorylation cascade systems.

The cyclic interconversion of enzymes between phosphorylated and unphosphorylated forms comprises a major mechanism of cellular regulation. A theoretical analysis of reversible covalent modification systems (Stadtman, E.R., and Chock, P.B. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2761-2765) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test qualitatively and quantitatively the theories and equations involved in this analysis, a model in vitro phosphorylation/dephosphorylation cyclic cascade was developed in which the converter enzymes catalyzing the covalent modifications were cAMP-dependent protein kinase (EC 2.7.1.37; type II) and phosphoprotein phosphatase (EC 3.1.3.16; Mr = 38,000), both purified to near homogeneity from bovine heart. The kinetic constants for both enzymes were fully characterized using the nanopeptide Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu as the interconvertible substrate, cAMP as an activator for the kinase, and Pi as an inhibitor for the phosphatase. In the presence of a nearly constant concentration of ATP, a steady-state level of phosphorylation of the peptide was attained which was determined by the relative concentrations of the kinase, phosphatase, and effectors. As predicted by the cyclic cascade model, this monocyclic cascade exhibited both signal amplification and an increase in sensitivity to variations in multiple effector concentrations. In addition, the data show that the steady-state level of phosphorylation obtained in the presence of an activator of the kinase (e.g. cAMP) and an inhibitor of the phosphatase (e.g. Pi) is a function of the product of the relative effector concentrations. Finally, the results reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than predicted previously.

Adenosine Triphosphate↗
PubMed1997

Incidentalomas. A disease of modern technology.

The optimal strategy for hormonal screening of a patient with any incidentally discovered adrenal or pituitary mass is unknown. Our review of the endocrinologic literature supports the view that such patients are at slightly increased risk for morbidity and mortality. There is a benefit of early diagnosis for at least for some of the disorders, suggesting the importance of case finding. The data in Tables 1 and 4 illustrate that clinically diagnosed hormone-secreting adrenal and pituitary tumors are far less common than incidentalomas. From a clinical perspective, our ability to determine accurately those at increased risk among the vast majority who are not at increased risk is poor. Given the limitations of diagnostic tests, effective hormonal screening requires a sufficiently high pretest probability to limit the number of false-positive results. This condition is met to varying degrees in the patient with an adrenal mass or small incidentally discovered pituitary mass but no signs or symptoms of hormone excess. Even the more common lesions such as pheochromocytoma and prolactinoma are relatively rare. Subjecting patients to unnecessary testing and treatment carries its own set of risks. Initial costs aside, testing may result in further expense and harm as false-positive results are pursued, producing the cascade effect described by Mold and Stein as a "chain of events (which) tends to proceed with increasing momentum, so that the further it progresses the more difficult it is to stop." The extensive evaluations performed in some patients with incidentally discovered masses may reflect the unwillingness of many physicians to accept uncertainty, even in the case of extremely unlikely diagnoses. This unwillingness may be driven, in part, by fear of potential malpractice liability, the failure to appreciate the influence of prevalence data on the interpretation of diagnostic testing, or other factors. Indeed, the major justification for further evaluation of these patients is not so much to avoid morbidity and mortality for rate patients who truly are at increased risk but rather to reassure those in whom further testing is negative (and to reassure ourselves). Physicians must take care not to create inappropriate anxiety in patients by overemphasizing the importance of an incidental finding unless it is associated with a realistic clinical risk. Our recommendations utilize currently available information to minimize the untoward effects of the cascade. As evidence accumulates, recommendations may need to be revised. The benefit of diagnosis of one of these adrenal or pituitary disorders must be considered in the context of the patient's overall condition. Studies are needed to analyze the utility in clinical practice of hormonal screening for these common radiologic findings. We need to use these studies to identify the critical gaps in our knowledge and to adopt the epidemiologic methods of evaluation of evidence that have been applied to preventive measures. We must be careful to recognize lead-time bias in which survival can seem to be lengthened when screening simply advances the time of diagnosis, lengthening the period of time between diagnosis and death without any true prolongation of life. Length bias refers to the tendency of screening to detect a disproportionate number of cases of slowly progressive disease and to miss aggressive cases that, by virtue of rapid progression, are present in the population only briefly. Endocrinologists must avoid the pitfalls of overestimation of disease prevalence and of the benefits of therapy resulting from advances in diagnostic imaging. Clinical judgment based on the best available evidence should be complemented and not replaced by laboratory data.

Adrenal Gland Diseases↗
PubMed2000

Pituitary incidentalomas.

The optimal strategy for hormonal screening of a patient with any incidentally discovered pituitary mass is unknown. The authors' review of the endocrinologic literature supports the view that such patients are at slightly increased risk for morbidity and mortality. This risk implies a benefit of early diagnosis for at least for some of the disorders, suggesting the importance of case finding. Nevertheless, the data in Table 1 illustrate that clinically diagnosed hormone-secreting pituitary tumors are far less common than incidentalomas. Clinically, one cannot accurately determine the approximately 0.5% of patients with incidentaloma who are at increased risk among the vast majority who are not. Given the limitations of diagnostic tests, effective hormonal screening requires a sufficiently high pretest probability to limit the number of false-positive results. This condition is met to varying degrees in the patient with a small incidentally discovered pituitary mass but no signs or symptoms of hormone excess. Even the more common lesions, such as prolactinoma, are relatively rare. [table: see text] Subjecting patients to unnecessary testing and treatment is associated with risk. In addition to its initial cost, testing may result in further expense and harm as false-positive results are pursued, producing the "cascade effect" described by Mold and Stein as a "chain of events (which) tends to proceed with increasing momentum, so that the further it progresses the more difficult it is to stop." The extensive evaluations performed for some patients with incidentally discovered masses may reflect the unwillingness of many physicians to accept uncertainty, even in the case of an extremely unlikely diagnosis. This unwillingness may be driven, in part, by fear of potential malpractice liability, the failure to appreciate the influence of prevalence data on the interpretation of diagnostic testing, or other factors. The major justification for further evaluation of these patients is not so much to avoid morbidity and mortality for the rare patient who truly is at increased risk but to reassure patients in whom further testing is negative and the physician. Physicians must take care not to create inappropriate anxiety in patients by overemphasizing the importance of an incidental finding unless it is associated with a realistic clinical risk. The authors' recommendations are based on currently available information to minimize the untoward effects of the cascade. As evidence accumulates, these recommendations may need to be revised. The benefit of the diagnosis of an adrenal or pituitary disorder must be considered in the context of the patient's overall condition. Additional studies are needed to analyze the clinical utility of hormonal screening for these common radiologic findings. Data from these studies can be used to identify critical gaps in knowledge and to adopt the epidemiologic methods of evaluation of evidence that have been applied to preventive measures. One must be careful to recognize lead-time bias, in which survival can appear to be lengthened when screening simply advances the time of diagnosis, lengthening the period of time between diagnosis and death without any true prolongation of life; and length bias, which refers to the tendency of screening to detect a disproportionate number of cases of slowly progressive disease and to miss aggressive cases that, by virtue of rapid progression, are present in the population only briefly. Physicians must avoid the pitfalls of overestimation of disease prevalence and of the benefits of therapy resulting from advances in diagnostic imaging. Clinical judgment based on the best available evidence should be complemented and not replaced by laboratory data.

Adenoma↗
PubMed1997

The development of a cascade impactor simulator based on adhesion force measurements to aid the development of dry powder inhalations.

Adhesion and friction forces are the main physical factors determining the re-suspension of a micronized drug from carrier particles during inhalation. Hence, it appears useful to link adhesion and friction force measurements to the in vitro testing of dry powder inhalations, namely the assessment of the mass median aerodynamic diameter (MMAD) using an eight-stage Andersen cascade impactor. Interactive mixtures of micronized Salmeterol Xinafoate adhered to irrespirable lactose monohydrate carrier particles were used as model dosage forms. The adhesion force between the drug and carrier particles was assessed using a centrifuge technique, and the MMAD was determined under standardized working conditions using the Andersen-Cascade impactor (Mark II). A cascade impactor simulator (CIS), which is a computer program containing a re-suspension model to assess the amount of drug detached from the carrier particles during inhalation, was developed and validated using the experimental data. It could be shown, that the CIS provided a good estimate of the loss of drug due to adhesion to the carrier particles and the loss of drug on the cascade impactor walls. Small deviations between the theoretical and experimental mass median aerodynamic particle diameters however were found. These deviations were shown to be mainly due to the experimental error introduced by the cascade impactor, and that the error due to the experimental adhesion measurements is negligibly small. Hence, the CIS developed could be a useful tool in early development stages of dry powder inhalations to predict the in vitro aerodynamic performance of drug particles.

Adhesiveness↗
PubMed1982

Acquired factor XI deficiency in systemic lupus erythematosus.

A variety of coagulation abnormalities has been described in systemic lupus erythematosus (SLE). We report a unique SLE patient with an acquired factor XI deficiency. The low factor XI levels were not due to a classical coagulation inhibitor, but to a plasma factor (probably an immunoglobulin) that selectively bound factor XI. The factor XI deficiency improved after treatment with corticosteroids. Thus it is possible that in SLE immune complexes may bind clotting factors making them unavailable for use in the coagulation cascade.

Blood Coagulation Tests↗
PubMed1998

Negative regulation of hrp genes in Pseudomonas syringae by HrpV.

Mutations in the five hrp and hrc genes in the hrpC operon of the phytopathogen Pseudomonas syringae pv. syringae 61 have different effects on bacterial interactions with host and nonhost plants. The hrcC gene within the hrpC operon encodes an outer membrane component of the Hrp secretion system that is conserved in all type III protein secretion systems and is required for most pathogenic phenotypes and for secretion of the HrpZ harpin to the bacterial milieu. The other four genes (in order), hrpF, hrpG, (hrcC), hrpT, and hrpV, appear to be unique to the group I hrp clusters found in certain phytopathogens (e.g., P. syringae and Erwinia amylovora) and are less well understood. We initiated an examination of their role in Hrp regulation and secretion by determining the effects of functionally nonpolar nptII cartridge insertions in each gene on the production and secretion of HrpZ, as determined by immunoblot analysis of cell fractions. P. syringae pv. syringae 61 hrpF, hrpG, and hrpT mutants were unable to secrete HrpZ, whereas the hrpV mutant overproduced and secreted the protein. This suggested that HrpV is a negative regulator of HrpZ production. Further immunoblot assays showed that the hrpV mutant produced higher levels of proteins encoded by all three of the major hrp operons tested-HrcJ (hrpZ operon), HrcC (hrpC operon), and HrcQB (hrpU operon)-and that constitutive expression of hrpV in trans abolished the production of each of these proteins. To determine the hierarchy of HrpV regulation in the P. syringae pv. syringae 61 positive regulatory cascade, which is composed of HrpRS (proteins homologous with sigma54-dependent promoter-enhancer-binding proteins) and HrpL (alternate sigma factor), we tested the ability of constitutively expressed hrpV to repress the activation of HrcJ production that normally accompanies constitutive expression of hrpL or hrpRS. No repression was observed, indicating that HrpV acts upstream of HrpRS in the cascade. The effect of HrpV levels on transcription of the hrpZ operon was determined by monitoring the levels of beta-glucuronidase produced by a hrpA'::uidA transcriptional fusion plasmid in different P. syringae pv. syringae 61 strains. The hrpV mutant produced higher levels of beta-glucuronidase than the wild type, a hrcU (type III secretion) mutant produced the same level as the wild type, and the strain constitutively expressing hrpV in trans produced low levels equivalent to that of a hrpS mutant. These results suggest that HrpF, HrpG, and HrpT are all components of the type III protein secretion system whereas HrpV is a negative regulator of transcription of the Hrp regulon.

Gene Expression Regulation, Bacterial↗
PubMed1999

The alternative "ischemic" cascade in coronary microvascular disease.

The conceptual model of the classical "ischemic cascade" has served cardiologists well for decades. It correctly predicts clinical findings during imaging stress testing in the presence of coronary artery disease or epicardial coronary artery spasm, where perfusion and wall motion abnormalities provide a substantially higher sensitivity than ECG changes. However, empirical experience has taught us that stress-induced ischemic-like ECG changes, often accompanied by perfusion abnormalities, are the rule rather than the exception in pathophysiological conditions during which the occurrence of ischemia usually cannot be proven, characterized by angiographically normal arteries and reduced flow reserve, such as syndrome X, arterial hypertension and hypertrophic cardiomyopathy. These stress-induced "echocardiographically silent" ST segment changes may be associated with impaired coronary flow reserve and systemic endothelial dysfunction. In hypertrophic cardiomyopathy stress-induced ischemic-like ST segment depression is linked to higher long-term incidence of adverse events. It is entirely likely that our monolithic view of ischemia mirrored in the classical ischemic cascade should be integrated by the awareness of the reverse or alternative "ischemic" cascade best describing microvascular disease, with ECG changes coming first, perfusion abnormalities second, and echocardiographic changes usually being absent. Not all forms of myocardial ischemia are the same, and milder, patchy degrees of myocardial ischemia--as those hypothesized, but not proven, in microvascular angina--remain silent in its mechanical functional manifestations and may well represent a physiological scotoma of stress echocardiography. "Anatomic lies" on the ECG may be overturned into "physiologic truths" when coronary flow reserve or systemic endothelial function is considered.

Coronary Circulation↗
PubMed2003

Validity of the allometric cascade model at submaximal and maximal metabolic rates in exercising men.

The dependence of metabolic rate (MR) on body mass (M) is described by the general allometric equation MR=aM(b), where, a is a proportionality coefficient and b is the mass exponent. Darveau et al. [Nature 417 (2002), 166] proposed a novel 'multiple-causes' allometric cascade model as a unifying principle of the scaling of MR, at rest and during maximal exercise. We tested the validity of body mass exponents predicted from the model for submaximal and maximal aerobic exercise conditions in 1629 men. MRs were estimated from whole-body oxygen consumption during an incremental treadmill test to voluntary exhaustion. For both submaximal (b=0.83) and maximal (b=0.94) exercise requiring average oxygen consumption rates of around 5-11 times resting values, respectively, the obtained mass exponents were remarkably consistent with predicted values. Moreover, for maximal MR the global mass exponent was significantly greater than for submaximal aerobic metabolism, congruent with the allometric cascade model.

Adult↗
PubMed2002

Inhibition of contact activation by a kininogen peptide (HKH20) derived from domain 5.

Contact activation can be initiated by interaction of Factor XII, prekallikrein (PK) and high molecular weight kininogen (HK) with inorganic negatively charged biologic macromolecules, or upon cell surfaces, or interaction with membrane protein derivatives such as aggregated beta amyloid. The latter two examples are zinc-dependent. The interaction with cells is dependent on peptides derived from HK domains 3 and 5 termed LDC27 and HKH20, respectively. We have tested the ability of each of these peptides to inhibit HK-dependent contact activation. HKH20 inhibited activation of prekallikrein when a mixture containing HK, prekallikrein and Factor XII was incubated with dextran sulfate, gC1qR, amyloid beta or endothelial cells. Comparable quantities of LDC27 had no effect. The binding of biotinylated HK or biotinylated Factor XII was inhibited in a dose response fashion by increasing concentrations of HKH20 while LDC27, again had no effect. The N-terminal region of HKH20 (amino acids 475-485) is of particular importance for binding and histidine 485 prominently enhances the reaction as assessed employing overlapping and deleted peptides. Since there is a role for HK heavy chain in binding to endothelial cells and LDC27 can be employed as an affinity ligand to isolate the binding proteins, we increased the LDC27 concentration from 10-fold to 250-fold to determine whether it is functional. Inhibition of endothelial cell-dependent prekallikrein activation required 100-fold greater concentration of LDC27 when compared to HKH20 to achieve significant inhibition. We conclude that the interactions of the light chain of HK via HKH20 is of particular importance for activation of the bradykinin forming cascade in zinc-dependent or independent reactions and is true for all "surface" initiators tested thus far.

Amino Acid Sequence↗
PubMed1999

Novel and known constituents from Buddleja species and their activity against leukocyte eicosanoid generation.

We have undertaken a systematic survey of the genus Buddleja used in traditional Chinese medicine for antiinflammatory and other indications by testing extracts and isolated natural products for their activity against the enzymes of the arachidonate cascade. This was done by using elicited rat peritoneal leukocytes, a physiologically relevant established whole cell system that expresses both cyclo-oxygenase (COX) and 5-lipoxygenase (5-LOX) activity. Lipophilic extracts of B. globosa roots and B. myriantha stem exhibited inhibitory activities in the 5-LOX and COX enzyme assays, whereas those of B. officinalis flowers, B. yunanesis stems, and B. asiatica stems showed inhibitory activities only against COX. The phytochemical investigation of these extracts, and consequent structure elucidation of isolated compounds using spectroscopic data, led to the isolation from B. globosa of three new terpenoid compounds named dihydrobuddledin A, buddledone A, and buddledone B and four known compounds-buddledins A, B, and C and zerumbone; 12 known compounds from B. officinalis-calceolarioside, campneoside, verbascoside, echinacoside, forsythoside B, angoroside A, crocetin monogentibiosyl ester, acacetin, acacetin-7-O-alpha-L-rhamnopyranosyl (1-6)-beta-D-glucopyranoside, acacetin-7-O-alpha-L-rhamnopyranosyl (1-6)[alpha-L-rhamnopyranosyl (1-2)]-beta-D-glucopyranoside, songarosaponin A, delta-amyrone; and eight known compounds fromB. yunanesis-11,14-dihydroxy-8,11, 13-abietatrien-7-one, beta-sitosterol, verbascoside, echinacoside, forsythoside B, angoroside A, methylcatapol, and sucrose. Tests on the isolated compounds for inhibition of eicosanoid synthesis showed that buddledin A, crocetin monogentibiosyl ester, and acacetin exhibited an inhibitory effect on COX with IC(50) values of 13.7 microM, 28.2 microM, and 77.5 microM, respectively, whereas buddledin A exhibited inhibitory effect on 5-LOX with an IC(50) value of 50.4 microM.

Animals↗
PubMed2001

Voltage-gated K(+) channel activity in human prostate cancer cell lines of markedly different metastatic potential: distinguishing characteristics of PC-3 and LNCaP cells.

BACKGROUND: Although ion channels are known to contribute to a variety of basic cellular behaviors involved in the metastatic cascade, their role in metastasis per se has only recently been questioned. The hypothesis tested was whether K(+) channels were different between strongly metastatic PC-3 and weakly metastatic LNCaP human prostate cancer cell lines. METHODS: The whole-cell configuration of the patch clamp recording technique was used to record voltage-gated currents from LNCaP and PC-3 cell lines. The responses to different voltage-clamp protocols, sensitivity to external Ca(2+), and addition of drugs and toxins were explored. RESULTS: Voltage-gated K(+) current density was significantly larger in LNCaP than PC-3 cells. In addition, the K(+) currents in a sub-population of PC-3 cells were Ca(2+)-sensitive. These properties reflected the differential metastatic character of the cells, the PC-3 cells appearing potentially more "excitable". CONCLUSIONS: Prostate cancer cells of varying metastatic ability can be distinguished by their ion channel characteristics. The possible contribution(s) of K(+) channel activity to development of malignancy needs exploration.

Calcium Channels↗
PubMed1999

Activation of nuclear factor-kappaB during orthotopic liver transplantation in rats is protective and does not require Kupffer cells.

Reperfusion after liver transplantation results in the induction of tumor necrosis factor-alpha (TNFalpha) as well as activation of the stress-associated signaling proteins, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1), and nuclear factor-kappaB (NF-kappaB). To test the hypothesis that Kupffer cells are involved in the activation of signal transduction cascades during rat liver transplantation, Kupffer cells were depleted from donor liver using gadolinium chloride (GdCl3), and then the activation of JNK, AP-1, and NF-kappaB were assessed after transplantation. The results showed that GdCl3 treatment did not inhibit the activation of these stress signals, although transplanted livers were depleted of Kupffer cells and partially protected from reperfusion injury. Interleukin-6 (IL-6) and IL-10 messenger RNAs (mRNAs) were induced by transplantation, and the induction was suppressed by Kupffer cell depletion. The induction of TNFalpha mRNA and serum protein during liver transplantation was unaffected by GdCl3. These results show that Kupffer cells are not a major source of TNFalpha production after liver transplantation and that stress-signaling protein activation occurs independently of Kupffer cells. Transplantation strongly activates the transcription factor NF-kappaB, which blocks TNFalpha-mediated apoptosis in hepatocytes in vitro. To assess the role of NF-kappaB activation during liver transplantation, the IkappaBalpha superrepressor was expressed in donor livers using adenoviral-mediated gene transfer. Inhibition of NF-kappaB resulted in increased serum alanine aminotransferase levels after 3 hours of transplantation. In addition, the blockade of NF-kappaB resulted in increased histological tissue injury and increased hepatic terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, indicating apoptosis. These results show that NF-kappaB activation has a protective role in the transplanted liver.

Alanine Transaminase↗
PubMed1996

Synthesis and in vitro reactivity of 3-carbamoyl-2-phenylpropionaldehyde and 2-phenylpropenal: putative reactive metabolites of felbamate.

We propose that 3-carbamoyl-2-phenylpropionaldehyde is an intermediate in the metabolism of felbamate, an anti-epileptic drug with a unique profile of the therapeutic activity, and undergoes a cascade of chemical reactions responsible for the toxic properties of the parent drug. To test this hypothesis, we have synthesized 3-carbamoyl-2-phenylpropionaldehyde and evaluated its in vitro reactivity. This molecule was found to be highly unstable at physiological pH (t1/2 < or = 30 s) and to undergo facile elimination to 2-phenylpropenal, an alpha, beta-unsaturated aldehyde commonly termed atropaldehyde. However, the predominant reaction pathway for 3-carbamoyl-2-phenylpropionaldehyde was reversible cyclization to generate 4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one, a urethane that has a considerably longer half-life at physiological pH (t1/2 > or = 5 h) and may serve as a stable reservoir of the reactive aldehyde both in vitro and in vivo. Atropaldehyde is a potent electrophile and was found to exhibit cytotoxicity to cultured fibroblasts (50% growth inhibition (GI50) = 4.1 +/- 1.1 microM) comparable to the known unsaturated aldehyde toxins, 4-hydroxy-2-nonenal and acrolein. 3-Carbamoyl-2-phenylpropionaldehyde also exhibited significant cytotoxicity (GI50 = 53 +/- 8 microM), whereas 2-phenyl-1,3-propanediol monocarbamate (GI50 > 500 microM) and 3-carbamoyl-2-phenylpropionic acid (GI50 > 500 microM) were nontoxic. We have additionally demonstrated the formation of a glutathione-atropaldehyde conjugate from the in vitro incubation of 3-carbamoyl-2-phenylpropionaldehyde with glutathione. Thus, the potent cytotoxicity and potential allergenicity of atropaldehyde implicate this unsaturated aldehyde as a possible causative agent in the toxicities observed with felbamate treatment.

Aldehydes↗
PubMed1998

Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation.

Activating point mutations in the Ras oncogene occur in a large number of human tumors, especially of epithelial origin. In thyroid follicular cells, ectopic expression of oncogenic H-Ras results in growth factor-independent proliferation, loss of differentiation and tumor formation in nude mice. In fibroblasts concomitant activation of the MAP kinase cascade and the small GTPase Rac-1 leads to full malignant transformation. We have tested the effects of these key downstream mediators of Ras in thyroid epithelial cells, by stably expressing either a constitutively active form of MEK-1 (MEK(deltaN3/S218E/S222D)), a constitutively active form of Rac-1 (Val12-Rac), or both. While the activation of one molecule or the other results in a weak phenotype, concomitant activation of both MEK-1 and Rac-1 in thyroid cells leads to growth factor-independent proliferation, morphological transformation and anchorage-independent growth. However, in contrast to Ras-transformed thyroid cells, the ones expressing the constitutively active forms of MEK-1 and Rac-1 maintain their differentiate phenotype and fail to form tumors when injected into nude mice. Thus, in thyroid epithelial cells, concomitant activation of MEK-1 and Rac-1 can reproduce only a subset of the Ras-induced effects and is not sufficient to cause full malignant transformation. Significantly, Ras-mediated increased proliferation and loss of differentiation can be dissociated in these cells.

Animals↗
PubMed1981

Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis.

Animals↗
PubMed1999

Systemic administration of NMDA and AMPA receptor antagonists reverses the neurochemical changes induced by nigrostriatal denervation in basal ganglia.

In Parkinson's disease, nigrostriatal denervation leads to an overactivity of the subthalamic nucleus and its target areas, which is responsible of the clinical manifestations of the disease. Because the subthalamic nucleus uses glutamate as neurotransmitter and is innervated by glutamatergic fibers, pharmacological blockade of glutamate transmission might be expected to restore the cascade of neurochemical changes induced by a dopaminergic denervation within the basal ganglia. To test this hypothesis, two types of glutamate antagonists, the NMDA receptor antagonist MK-801 and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist LY293558, were administered systemically, either alone or in combination with L-DOPA, in rats with a unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway. The effect of treatment was assessed neurochemically by analyzing at the cellular level the functional activity of basal ganglia output structures and the subthalamic nucleus using the expression levels of the mRNAs coding for glutamic acid decarboxylase and cytochrome oxidase, respectively, as molecular markers of neuronal activity. The present study shows that treatment with glutamate antagonists, and particularly with AMPA antagonists, alone or in combination with L-DOPA, reverses the overactivity of the subthalamic nucleus and its target areas induced by nigrostriatal denervation. These results furnish the neurochemical basis for the potential use of glutamate antagonists as therapeutic agents in Parkinson's disease.

Animals↗
PubMed2002

Insulin-like growth factor I induces MDM2-dependent degradation of p53 via the p38 MAPK pathway in response to DNA damage.

In many tissues, the insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to functionally oppose apoptosis. Recently, we demonstrated a direct role for the IGF-IR in the rescue of DNA-damaged fibroblasts by activating a DNA repair pathway (Héron-Milhavet, L., Karas, M., Goldsmith, C. M., Baum, B. J., and LeRoith, D. (2001) J. Biol. Chem. 276, 18185-18192). p53 is a nuclear transcription factor that can block progression of the cell cycle, modulate DNA repair, and trigger apoptosis. In this work, we tested the effect of IGF-I on the regulation of the p53 signaling cascade. The DNA-damaging agent 4-nitroquinoline 1-oxide was applied to NIH-3T3 cells overexpressing normal IGF-IRs (NWTb3 cells). We showed that after 4-nitroquinoline 1-oxide-induced DNA damage, IGF-I induced exclusion of the p53 protein from the nucleus and led to its degradation in the cytoplasm, whereas p53 mRNA was unaffected. Degradation of the p53 protein was associated with an increase in MDM2, an upstream modulator of the half-life and activity of the p53 protein. p53 degradation was also associated with down-regulation of p21. We further showed that the effects of IGF-I on mdm2 transcription and on MDM2/p19 ARF association were mediated by the p38 MAPK pathway. In conclusion, we describe a novel role for IGF-I in the regulation of the MDM2/p53/p21 signaling pathway during DNA damage.

Cell Line↗
PubMed1999

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.

Activin Receptors, Type I↗