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PubMed1998

Marked reduction in internal atrial defibrillation thresholds with dual-current pathways and sequential shocks in humans.

BACKGROUND: This study tested the ability of sequential shocks delivered through dual-current pathways to lower the atrial defibrillation threshold (ADFT) compared with a biphasic shock through a standard single-current pathway. METHODS AND RESULTS: Electrodes were positioned in the right atrial appendage (RA), left subclavian vein (LSV), proximal coronary sinus (CSos), and distal coronary sinus (DCS) in 14 patients with chronic atrial fibrillation (170+/-185 days). Using a step-up protocol, we compared ADFTs for a single-current pathway (RA-->DCS) that used a single 7.5/2.5-ms biphasic shock from a 150-microF capacitor with those for a dual-current pathway system (RA-->DCS followed by CSos-->LSV) using sequential 7.5/2.5-ms biphasic shocks with capacitor discharge waveforms for 150-microF and 600-microF capacitors. Both dual-current pathway configurations (2.0+/-0.4 J for 150-microF capacitance, 2.4+/-0.5 J for 600-microF capacitance) had a significantly lower ADFT than the single-current pathway (5.1+/-1.8 J). Whereas the dual-current pathway with 150-microF capacitor shocks had a significantly lower energy threshold, there was no statistical difference in terms of leading-edge voltage compared with the dual-current pathway with 600-microF capacitance shocks. There were no ventricular arrhythmias induced with appropriately synchronized shocks. CONCLUSIONS: For internal atrial defibrillation in humans, sequential biphasic waveforms delivered over dual-current pathways resulted in a markedly reduced (>50% reduction) ADFT compared with a single shock over a single-current pathway.

Adult↗
PubMed2005

Genetic dissection of the signaling pathways that control gastric acid secretion.

Gastric acid secretion is regulated by endocrine, paracrine and neurocrine signals via at least three pathways, the gastrin-histamine pathway, the CCK-somatostatin pathway and the neural pathway. Genetically-engineered mice, subjected to targeted gene disruption (i.e., knockout mice), have been used to dissect the signaling pathways that are responsible for the complexity of the regulation of acid secretion in vivo. Both gastrin knockout and gastrin/CCK2 receptor knockout mice displayed greatly impaired acid secretion, presumably because of the loss of the gastrin-histamine pathway. Gastrin/CCK double-knockout mice had a relatively high percentage of active parietal cells with a maintained ability to respond with copious acid secretion to pylorus ligation-evoked vagal stimulation and to a histamine challenge. The low acid secretion in gastrin knockout mice and gastrin/CCK2 receptor knockout mice and the restoration of acid secretion in gastrin/CCK double-knockout mice suggest that CCK plays an important role as inhibitor of the parietal cells via the CCK-somatostatin pathway by stimulating the CCK1 receptor of the D cell. In the absence of both the gastrin-histamine and the CCK-somatostatin pathway (as in gastrin/CCK2 receptor double-knockout mice), the control of acid secretion is probably taken over by neural pathways, explaining the high acid output. The observations illustrate the complexity and plasticity of the acid regulatory mechanisms. It seems that one pathway may be suppressed or allowed to dominate over the others depending on the circumstances.

Animals↗
PubMed2006

Identification of genes regulated by Wnt/beta-catenin pathway and involved in apoptosis via microarray analysis.

BACKGROUND: Wnt/beta-catenin pathway has critical roles in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of this pathway, little is known regarding Wnt/beta-catenin pathway modification of the cellular apoptosis. METHODS: To identify potential genes regulated by Wnt/beta-catenin pathway and involved in apoptosis, we used a stably integrated, inducible RNA interference (RNAi) vector to specific inhibit the expression and the transcriptional activity of beta-catenin in HeLa cells. Meanwhile, we designed an oligonucleotide microarray covering 1384 apoptosis-related genes. Using oligonucleotide microarrays, a series of differential expression of genes was identified and further confirmed by RT-PCR. RESULTS: Stably integrated inducible RNAi vector could effectively suppress beta-catenin expression and the transcriptional activity of beta-catenin/TCF. Meanwhile, depletion of beta-catenin in this manner made the cells more sensitive to apoptosis. 130 genes involved in some important cell-apoptotic pathways, such as PTEN-PI3K-AKT pathway, NF-kappaB pathway and p53 pathway, showed significant alteration in their expression level after the knockdown of beta-catenin. CONCLUSION: Coupling RNAi knockdown with microarray and RT-PCR analyses proves to be a versatile strategy for identifying genes regulated by Wnt/beta-catenin pathway and for a better understanding the role of this pathway in apoptosis. Some of the identified beta-catenin/TCF directed or indirected target genes may represent excellent targets to limit tumor growth.

Apoptosis↗
PubMed1975

The choline incorporation pathway: primary mechanism for de novo lecithin synthesis in fetal primate lung.

The two pathways of de novo lecithin synthesis, choline incorporation (I) and phosphatidylethanolamine methylation (II), were examined in lung slices from rhesus monkey fetuses throughout the last half of gestation. Incorporation rates of pathway-specific radioactive precursors were used as a measure of lecithin production. At all stages of development studied, pathway I incorporated 10-50 times more precursor ([14C]choline) into lecithin than did pathway II ([14C]methionine or [14C]ethanolamine). In addition, although methylation activity did not change significantly during gestation, choline incorporation showed three distinct phases: (1) a stable, relatively low rate in early gestation, (2) an abrupt, twofold increase at approximately 90 percent of term, and (3) a return to lower activity levels in late gestation. This correlates with reports that lung lecithin concentration in fetal primates increases significantly in the last 10 percent of gestation. The lecithin to spingomyelin (L/S) ratios measured in amniotic fluid samples obtained at abdominal delivery were compared with pathway activities in lung slices from the same fetuses. Significant correlation was found between the amniotic fluid L/S ratio and pathway I activity (r = 0.77, P less than 0.001); in contrast, pathway II activity showed no relationship to the amniotic fluid L/S ratio. Thus, the L/S ratio appears to be a reflection of lung lecithin synthesis through the choline pathway. The conclusion that the choline pathway is the primary route of de novo lecithin synthesis in the nonhuman fetal primate lung is supported by three lines of evidence, (1) the predominance of choline incorporation into lecithin, (2) the late gestational rise in conversion of choline to lecithin, and (3) the correlation between pathway I activity and both lung lecithin concentration and amniotic fluid L/S ratio.

Adenosine Triphosphate↗
PubMed1997

Detection of the three Kunitz-type single domains of membrane-bound tissue factor pathway inhibitor (TFPI) by flow cytometry.

Tissue factor pathway inhibitor, a natural anticoagulant in the extrinsic pathway of blood coagulation, is associated with the endothelial membrane and presumed to be released by heparin. For flow cytometric detection of membrane-bound tissue factor pathway inhibitor we synthesized polyclonal monospecific antibodies directed against each of the three Kunitz-type domains. Antisera were obtained by immunisation of rabbits with synthetic oligopeptides representing the reactive site of each domain. Kunitz-domain delta 1: 26CAFKDDGPCKAIMKR41, domain delta 2: 101EDPGICRGYITR112 and domain delta 3: 192PADRGLCRANENR204. Different cell lines (chondrosarcoma, synovial sarcoma, synovial cells, leukaemic monocytes) and endothelial cells were investigated by flow cytometric analysis using these antibodies. The three tissue factor pathway inhibitor domains were detected on the surface of all cells by the corresponding antisera. Similar results were obtained by immuno-histochemical staining. Since domain delta 3 was recognised by the appropriate antibody, it would seem that this third domain is not the membrane binding site. To investigate the cellular tissue factor pathway inhibitor release, endothelial cells were cultivated with heparin. Protein resynthesis and translocation were inhibited by puromycin and monensin, respectively. After heparin incubation an increased tissue factor pathway inhibitor concentration was determined in the cell culture medium by a chromogenic substrate assay. However, the tissue factor pathway inhibitor density on the cell surface was not influenced by heparin, as shown by flow cytometry using the three tissue factor pathway inhibitor antisera. Our results suggest that functionally active tissue factor pathway inhibitor is not released from the cell surface. Therefore, the effect of heparin appears to be mediated by secretion of tissue factor pathway inhibitor from intracellular stores.

Amino Acid Sequence↗
PubMed2004

Convergent, RIC-8-dependent Galpha signaling pathways in the Caenorhabditis elegans synaptic signaling network.

We used gain-of-function and null synaptic signaling network mutants to investigate the relationship of the G alpha(q) and G alpha(s) pathways to synaptic vesicle priming and to each other. Genetic epistasis studies using G alpha(q) gain-of-function and null mutations, along with a mutation that blocks synaptic vesicle priming and the synaptic vesicle priming stimulator phorbol ester, suggest that the G alpha(q) pathway generates the core, obligatory signals for synaptic vesicle priming. In contrast, the G alpha(s) pathway is not required for the core priming function, because steady-state levels of neurotransmitter release are not significantly altered in animals lacking a neuronal G alpha(s) pathway, even though these animals are strongly paralyzed as a result of functional (nondevelopmental) defects. However, our genetic analysis indicates that these two functionally distinct pathways converge and that they do so downstream of DAG production. Further linking the two pathways, our epistasis analysis of a ric-8 null mutant suggests that RIC-8 (a receptor-independent G alpha guanine nucleotide exchange factor) is required to maintain both the G alpha(q) vesicle priming pathway and the neuronal G alpha(s) pathway in a functional state. We propose that the neuronal G alpha(s) pathway transduces critical positional information onto the core G alpha(q) pathway to stabilize the priming of selected synapses that are optimal for locomotion.

Animals↗
PubMed2004

Mutations that rescue the paralysis of Caenorhabditis elegans ric-8 (synembryn) mutants activate the G alpha(s) pathway and define a third major branch of the synaptic signaling network.

To identify hypothesized missing components of the synaptic G alpha(o)-G alpha(q) signaling network, which tightly regulates neurotransmitter release, we undertook two large forward genetic screens in the model organism C. elegans and focused first on mutations that strongly rescue the paralysis of ric-8(md303) reduction-of-function mutants, previously shown to be defective in G alpha(q) pathway activation. Through high-resolution mapping followed by sequence analysis, we show that these mutations affect four genes. Two activate the G alpha(q) pathway through gain-of-function mutations in G alpha(q); however, all of the remaining mutations activate components of the G alpha(s) pathway, including G alpha(s), adenylyl cyclase, and protein kinase A. Pharmacological assays suggest that the G alpha(s) pathway-activating mutations increase steady-state neurotransmitter release, and the strongly impaired neurotransmitter release of ric-8(md303) mutants is rescued to greater than wild-type levels by the strongest G alpha(s) pathway activating mutations. Using transgene induction studies, we show that activating the G alpha(s) pathway in adult animals rapidly induces hyperactive locomotion and rapidly rescues the paralysis of the ric-8 mutant. Using cell-specific promoters we show that neuronal, but not muscle, G alpha(s) pathway activation is sufficient to rescue ric-8(md303)'s paralysis. Our results appear to link RIC-8 (synembryn) and a third major G alpha pathway, the G alpha(s) pathway, with the previously discovered G alpha(o) and G alpha(q) pathways of the synaptic signaling network.

Adenylyl Cyclases↗
PubMed1992

Safety and efficacy of a modified catheter-mediated ablation of accessory pathways.

Fifty-one consecutive patients underwent modified catheter-mediated direct-current ablation of accessory pathways. Energy was delivered through the distal pair of electrodes (dual electrode configuration) of a 6F quadripolar catheter to the internal surface of the right or left atrioventricular (AV) annulus. In an attempt to prevent the later resumption of accessory pathway conduction, one additional shock was given after the initial successful interruption of accessory pathways. A mean of 2.7 shocks with cumulative energy of 453 +/- 32 Joules/patient interrupted the accessory pathways in 47 patients and modified the accessory pathway conduction in 2 patients. Forty-eight patients were asymptomatic and free of any antiarrhythmic agents with a follow-up ranging from 3-20 months (mean 12 +/- 1 months), without early or late serious complications (AV block or tamponade). Conduction characteristics, concealed or manifest, and recording of accessory pathway activity did not affect the outcome. Mean cumulative energy and number of applications of energy to achieve a successful outcome were lower in patients with concealed (376 +/- 31 Joules, 2.4 +/- 0.2 shocks) than manifest accessory pathways (516 +/- 50 Joules, 2.9 +/- 0.2 shocks). At the successful ablation sites, the mean shortest retrograde ventriculoatrial interval during orthodromic reentrant tachycardia (VA') was 80 +/- 3 msec (78% had VA' less than 90 msec) and was not different between concealed and manifest accessory pathways; the mean shortest antegrade AV interval was 47 +/- 3 msec in manifest preexcitation; the mean ratio of atrial to ventricular wave amplitude was not significantly different between left-sided (0.8 +/- 0.1) and right-sided (1.1 +/- 0.2) accessory pathways (p greater than 0.05). A successful outcome was achieved in 94% of 51 patients. This procedure is relatively safe and effective, regardless of the location of the accessory pathway.

Adult↗
PubMed1994

Thalamic ventrobasal stimulation for pain relief. Probable mechanisms, pathways and neurotransmitters.

Thalamic ventrobasal (VB) stimulation, first performed by Mazars, in 1961, is a valuable means for treating central and deafferentation pain. The way it acts to achieve pain relief, however, is still a matter of controversy. In this paper, the author examines previously proposed hypotheses and suggests that VB stimulation induces pain relief by activation of a multisynaptic inhibitory pathway to the medial thalamus, in which the dopaminergic nigrostriatal system exerts an important role and by modulation of abnormal activity in VB itself. The multisynaptic pathway involved, as well as the neurotransmitters, are suggested: VB stimulation excites somatosensory cortex through the glutaminergic thalamocortical pathway, which in turn, sends excitatory glutaminergic axons to the motor cortex. The sensorymotor cortex originates the excitatory glutaminergic corticostriatal pathway to the anterior putamen. The anterior putamen sends excitatory peptidergic (substance P) pathways to the globus pallidus internus (striatopallidal pathway) and to the substantia nigra reticulata (striatonigral pathway). The globus pallidus internus inhibits the medial thalamus through the pallidothalamic GABAergic pathway. The substantia nigra reticulata sends inhibitory GABAergic projections to the medial thalamus (nigrothalamic pathway) and excites the substantia nigra compacta. The substantia nigra compacta projects excitatory dopaminergic axons to the striatal neurons (nigrostriatal pathway) with output to the globus pallidus internus and substantia nigra reticulata and so on. Data to support this hypothesis are provided by an extensive review of the literature.

Analgesia↗
PubMed2006

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects.

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.

Bystander Effect↗
PubMed2004

PathAligner: metabolic pathway retrieval and alignment.

MOTIVATION: Analysis of metabolic pathways is a central topic in understanding the relationship between genotype and phenotype. The rapid accumulation of biological data provides the possibility of studying metabolic pathways at both the genomic and the metabolic levels. Retrieving metabolic pathways from current biological data sources, reconstructing metabolic pathways from rudimentary pathway components, and aligning metabolic pathways with each other are major tasks. Our motivation was to develop a conceptual framework and computational system that allows the retrieval of metabolic pathway information and the processing of alignments to reveal the similarities between metabolic pathways. RESULTS: PathAligner extracts metabolic information from biological databases via the Internet and builds metabolic pathways with data sources of genes, sequences, enzymes, metabolites etc. It provides an easy-to-use interface to retrieve, display and manipulate metabolic information. PathAligner also provides an alignment method to compare the similarity between metabolic pathways. AVAILABILITY: PathAligner is available at http://bibiserv.techfak.uni-bielefeld.de/pathaligner.

Algorithms↗
PubMed2004

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses.

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.

CD40 Ligand↗
PubMed2000

[Clinical pathways in oncology].

The diagnosis-related-group/prospective-payment system (DRG/PPS) was introduced into the health care system of the United States in 1983. This system triggered the development and implementation of clinical pathways aimed at reducing the length and cost of hospitalization. In Japan, trial use of a Japanese version of DRG/PPS was initiated in November 1998 in 10 hospitals under the control of the Ministry of Health and Welfare, and full-scale implementation of the system is expected in the near future. Clinical pathways, therefore, are a current focus of attention, mainly because of their success in enhancing management efficiency in the U.S. However, in actual clinical settings where clinical pathways are used, several Japanese health care providers have come to realize that they are also useful in improving staff coordination, patient satisfaction, and patient care, rather than simply reducing the length of hospital stay and cost of health care. The introduction of clinical pathways requires that treatment of the disease in question be defined and standardized. The implementation of pathways for the treatment of cancer, however, might prove difficult because of the high frequency of variation. In our experience, the main reason for the use of clinical pathways is not to reduce the number of variant cases but to provide high-quality care through the promotion of a team approach to treatment and enhanced patient care. Therefore, even if there were frequent variances following surgery for cancer, those occurring in accordance with the pathophysiological state of the patient would not interfere with management by clinical pathways. Clinical pathways are advantageous because they allow patients to know their treatment schedule; to prepare for hospitalization procedures; to have a better perspective on discharge; to reduce anxiety regarding hospital admission, even if it is the first time; to communicate better with doctors, nurses, and other medical care staff, leading to greater trust; and to improve their ability for self-management. These features are all important for the improvement of patient care. Furthermore, clinical pathways may lead to a situation in which the cost of hospitalization can be predicted prior to admission, enabling patients to compare differences between several hospitals. From our experience with gastric cancer, breast cancer, and esophageal cancer management, we consider clinical pathways to be of great benefit in helping to reform the current medical care system in regard to the management of cancer patients as well as patients with other diseases.

Breast Neoplasms↗
PubMed2004

Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells.

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed1992

[Catheter ablation of septal accessory conduction pathways with high frequency electric current].

In 58 symptomatic patients with septal accessory atrioventricular pathways, attempts at catheter ablation of the pathway were made using 500-kHz radiofrequency current. The methodological approach (introduction and final positioning of the ablation catheter) was dependent on the anatomical site of the accessory pathway. Right anteroseptal pathways were accessed via a jugular venous route, whereas a femoral venous route was used for right mid- and posteroseptal pathways. In these pathways, ablation was attempted from an atrial catheter position. Left posteroseptal pathways were located via mapping of the coronary sinus and were ablated either from the left ventricle or (in 3 cases) from the vena cordis media. Utilizing a deflectable catheter with a 4-mm tip electrode, ablation attempts were successful in 54 patients (93%) with a median of 12 radiofrequency current pulses of an average 24.9 W of power and 23.2 s length. The mean duration of the sessions was 4.6 h. Impairment of physiological conduction (first-degree AV block) was observed in 1 patient; complete heart block was never induced. Recurrences after initially successful ablation necessitated a repeat session in 2 patients. One patient died 3 days after successful ablation of a posteroseptal accessory pathway. Septal accessory pathways may be ablated using radiofrequency current with an efficacy and safety comparable to free-wall accessory pathways and with good preservation of physiological AV node-His bundle conduction.

Adolescent↗
PubMed2007

An imbalance between Smad and MAPK pathways is responsible for TGF-beta tumor promoting effects in high-grade gliomas.

The transforming growth factor-beta (TGF-beta) plays a pivotal role in the pathobiology of human gliomas: during carcinogenesis, it turns from a tumor suppressor to a tumor promoter. The traditional Smad pathway and the more recently discovered MAPK pathway are the most important pathways for TGF-beta related intracellular signal transduction mediating differential pathobiological effects. In this study, we investigated the effects of TGF-beta2 and the TGF-beta2 antisense phosphorothioate oligodeoxynucleotide (PTO) AS-11 on the functionality of both the Smad and MAPK pathways in high-grade gliomas. We aimed to correlate the imbalance between the pathways with differences in the behaviour of high-grade glioma cells. Gene and protein expression studies were used to detect levels of members of the Smad and MAPK pathways under regulation of TGF-beta2 and AS-11. Proliferation and migration assays were functional readouts for effects caused by these regulating tools. Gene arrays were used to detect yet unknown regulators of these functional effects. The Smad pathway was functional in the tested cell lines. Exogenous TGF-beta2 inhibited proliferation but enhanced migration. Smad 2 mRNA expression and activation were significantly reduced by incubation with AS-11. K-ras was reduced both in gene arrays and quPCR under treatment with AS-11, but there was no influence of K-ras down-regulation on the activity of ERK. Ubiquitination-related genes also were specifically down-regulated with AS-11. Our results indicate the involvement of K-ras in TGF-beta signaling in high-grade gliomas. ERK, which is a member of the MAPK pathway, was not influenced and seems to be activated through RAS independent cascades in glioma. These results suggest that combined antagonization of the TGF-beta and MAPK pathways might be a promising approach for glioma therapy. An imbalance between these two pathways might be responsible for TGF-beta switching to a tumor promoter protein in high-grade gliomas.

Brain Neoplasms↗
PubMed1977

Pathways of complement activation in membranoproliferative glomerulonephritis and allograft rejection.

The complement system is comprised of at least 18 plasma proteins and consists of four functional divisions: two pathways for activation (classical and alternative), a common amplification mechanism for the activating pathways, and a final common effector pathway to which the activating and amplifying sequences are directed. The classical pathway is activated by certain antigen-antibody complexes, while the alternative pathway may be initiated non-immunologically by various microbial polysaccharides. Indeed, mixtures of purified C3, B, D, and P regulated to low-grade interaction by the presence of C3bINA and beta1 H respond to zymosan with amplified C3 and B inactivation. Both pathways form enzymes termed C3 convertases that cleave C3 to generate its major fragment, C3b. C3b interacts with each C3 convertase to permit C5 cleavage in activation of the effector complement sequence, and it interacts with alternative-pathway factors B and D to generate additional C3 convertase, C3bBb, in the amplification pathway. As C3 cleavage represents the most critical step in the elaboration of the biologic effects of the complement system, modulation of this reaction by generation, stabilization, and inactivation of the amplification convertase C3bBb may well determine whether initial activation of the complement sequence eventuates in beneficial or detrimental effects for the host. Initial generation of C3bBb is dependent on prior cleavage of C3, which may occur by the classical pathway or the alternative pathway. Stabilization of C3bBb is achieved with either P or C3NeF after their binding to C3b and C3bBb, respectively. Control of this amplifying step occurs at three levels: intrinsic decay of the inherently labile C3bBb complex, extrinsic decay-dissociation of Bb from the complex by beta1H, and inactivation of C3b by C3bINA. In the presence of stabilizing factors the control proteins must function in sequence, since C3bINA cannot act on C3bBb; beta1H-mediated decay of protective Bb must precede C3b inactivation by C3bINA. C3NeF, which is found in the sera of some patients with MPGN and persistent depressions of serum C3, circumvents all three controls because of its capacity to create a stabilized convertase that is relatively resistant to decay-dissociation by beta1H. The effector complement sequence is activated by cleavage of C3 and C5, which releases vasoactive and chemotactic peptides, C3a and C5a, and generates the major fragments C3b and C5b. C3b, in addition to its function in the amplifying reaction and the C5 convertases, mediates immune adherence to cells possessing membrane-associated receptors for C3b; this in turn promotes the phagocytic and secretory functions unique to each cell type. Cell-bound C5b serves to assemble the cytolytic complex C5b6789, while fluid-phase C5d generates the hemolytically inactive chemotactic complex C567d...

Complement C3↗
PubMed1982

Identification of a new C-23 oxidation pathway of metabolism for 1,25-dihydroxyvitamin D3 present in intestine and kidney.

Evidence is presented for the existence of a new C-23 oxidation pathway for the metabolism of the hormonally active form of vitamin D3, namely 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Homogenates of intestinal mucosa or kidney, but not liver, from rats and chicks convert 1,25(OH)2[26,27-3H]D3 into two new metabolites, 1 alpha,25-dihydroxy-23-oxo-vitamin D3 (1,25(OH)2-23-oxo-D3) and 1 alpha,25,26-trihydroxy-23-oxo-vitamin D3 (1,25,26(OH)3-23-oxo-D3), and an unknown metabolite(s) which has been possibly subjected to side chain modification/cleavage so that the tritium of the substrate has been converted into a form which is volatile. The isolation and chemical characterization of 1,25(OH)2-23-oxo-D3 and 1,25,26(OH)3-23-oxo-D3 from homogenates of chick intestinal mucosa has recently been described (Ohnuma, N., Kruse, J., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). Based on kinetic studies with chick intestinal homogenates, the proposed C-23 oxidation pathway is: 1,25(OH)2D3 leads to 1,25(OH)2-23-oxo-D3 leads to 1,25,26(OH)3-23-oxo-D3 leads to unknown metabolite with altered side chain. The relative enzyme activities of the C-23 pathway in tissues from vitamin D-replete chicks are: intestine, kidney liver, 18:3:less than 1. The activity of the C-23 pathway in homogenates of chick intestinal mucosa can be enhanced 10 x by prior priming of the birds with a single intravenous dose of 500 ng (1.3 nmol) of 1,25(OH)2D3; the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels by 12 h. A comparison was made in chick and rat intestinal homogenates of this C-23 pathway and the previously known C-24 oxidation pathway, which converts 1,25(OH)2D3 into 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3); it was calculated that under these conditions 72% of the 1,25(OH)2D3 was metabolized by the C-23 oxidation pathway, 13% by the C-24 pathway, and only 14% by other as yet unspecified pathways. It is proposed that the newly discovered C-23 pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney may play a prominent role under physiological conditions of controlling the tissue levels of this hormonally active form of vitamin D3.

Animals↗