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PubMed2002

Rapid kinetic studies of acetyl-CoA synthesis: evidence supporting the catalytic intermediacy of a paramagnetic NiFeC species in the autotrophic Wood-Ljungdahl pathway.

CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO(2) fixation, is a bifunctional enzyme containing CODH, which catalyzes the reversible two-electron oxidation of CO to CO(2), and ACS, which catalyzes acetyl-CoA synthesis from CoA, CO, and a methylated corrinoid iron-sulfur protein (CFeSP). ACS contains an active site nickel iron-sulfur cluster that forms a paramagnetic adduct with CO, called the nickel iron carbon (NiFeC) species, which we have hypothesized to be a key intermediate in acetyl-CoA synthesis. This hypothesis has been controversial. Here we report the results of steady-state kinetic experiments; stopped-flow and rapid freeze-quench transient kinetic studies; and kinetic simulations that directly test this hypothesis. Our results show that formation of the NiFeC intermediate occurs at approximately the same rate as, and its decay occurs 6-fold faster than, the rate of acetyl-CoA synthesis. Kinetic simulations of the steady-state and transient kinetic results accommodate the NiFeC species in the mechanism and define the rate constants for the elementary steps in acetyl-CoA synthesis. The combined results strongly support the kinetic competence of the NiFeC species in the Wood-Ljungdahl pathway. The results also imply that the methylation of ACS occurs by attack of the Ni(1+) site in the NiFeC intermediate on the methyl group of the methylated CFeSP. Our results indicate that CO inhibits acetyl-CoA synthesis by inhibiting this methyl transfer reaction. Under noninhibitory CO concentrations (below 100 microM), formation of the NiFeC species is rate-limiting, while at higher inhibitory CO concentrations, methyl transfer to ACS becomes rate-limiting.

Acetate-CoA Ligase↗

PubMed2004

Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells.

Gene expression was analyzed in five pairs of 5-fluorouracil (5-FU) resistant and parental cancer cell lines on DNA microarrays. In unsupervised analysis, a prediction rule was built from the expression profiles of 29 genes, and 5-FU sensitivity class was predicted with 100% accuracy and high predictive strength. In supervised analysis of key 5-FU pathways, expression of 91 genes was associated with 5-FU sensitivity phenotype and segregated samples accordingly in hierarchical analysis. Key genes involved in 5-FU activation were significantly down-regulated (thymidine kinase, 2.9-fold; orotate phosphoribosyltransferase, 2.3-fold; uridine monophosphate kinase, 3.2-fold; pyrimidine nucleoside phosphorylase 3.6-fold) in resistant cells. Overexpression of thymidylate synthase and its adjacent gene, c-Yes, was detected in the resistant cell lines. The mRNA and protein overexpression of nuclear factor kappaB (NFkappaB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFkappaB DNA-binding activity. Cotransfection of NFkappaB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFkappaB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G(1)-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G(1), and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance.

Base Sequence↗

PubMed2005

Muscle-specific Pten deletion protects against insulin resistance and diabetes.

Pten (phosphatase with tensin homology), a dual-specificity phosphatase, is a negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Pten regulates a vast array of biological functions including growth, metabolism, and longevity. Although the PI3K/Akt pathway is a key determinant of the insulin-dependent increase in glucose uptake into muscle and adipose cells, the contribution of this pathway in muscle to whole-body glucose homeostasis is unclear. Here we show that muscle-specific deletion of Pten protected mice from insulin resistance and diabetes caused by high-fat feeding. Deletion of muscle Pten resulted in enhanced insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in soleus but, surprisingly, not in extensor digitorum longus muscle compared to littermate controls upon high-fat feeding, and these mice were spared from developing hyperinsulinemia and islet hyperplasia. Muscle Pten may be a potential target for treatment or prevention of insulin resistance and diabetes.

Animals↗

PubMed2003

The GTP/GDP cycling of rho GTPase TCL is an essential regulator of the early endocytic pathway.

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1-positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1-positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.

Cell Membrane↗

PubMed2004

The adaptor protein Nck1 mediates endothelin A receptor-regulated cell migration through the Cdc42-dependent c-Jun N-terminal kinase pathway.

Cell migration plays key roles in physiological and pathological phenomena, such as development and oncogenesis. The adaptor proteins Grb2, CrkII, and Nck1 are composed of only a single Src homology 2 domain and some Src homology 3 domains, giving specificity to each signal transduction pathway. However, little is known about the relationships between their adaptor proteins and cell migration, which are regulated by the G protein-coupled receptor. Here we showed that Nck1, but not Grb2 or CrkII, mediated the inhibition of cell migration induced by the endothelin-1 and endothelin A receptor. The small interference RNA and dominant negative mutants of Nck1 diminished the endothelin-1-induced inhibition of cell migration. Although overexpression of wild-type Nck1 was detected in the cytosol and did not affect cell migration, expression of the myristoylation signal sequence-conjugated Nck1 was detected in the membrane and induced activation of Cdc42 and c-Jun N-terminal kinase, inhibiting cell migration. Taken together, these results suggest that the endothelin A receptor transduces the signal of inhibition of cell migration through Cdc42-dependent c-Jun N-terminal kinase activation by using Nck1.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A widespread and early requirement for a novel Notch function during Drosophila embryogenesis.

The Notch pathway plays a key role in the formation of many tissues and cell types in Metazoans. We recently showed that Notch acts in two pathways to determine muscle precursor fates. The first is the "standard" Notch pathway, in which Delta activates the Notch receptor, which then translocates into the nucleus in conjunction with Su(H) to reprogram transcription patterns and bring about changes in cell fates. The second pathway is poorly defined, but known to be independent of the ligands and downstream effectors of the standard pathway. The standard pathway is required in many different developmental contexts and we wondered if there was also a general requirement for the novel pathway. Here we show that the novel Notch pathway is required for the development of each of five examined cell types. These results indicate that the novel pathway is a widespread and fundamental component of Notch function. We further show that both Notch pathways operate in the differentiation of the same cell types. In such cases, the novel pathway acts first and appears to set up or limit the size of equivalence groups. The standard pathway then acts within the equivalence groups to limit individual cell fates.

Animals↗

PubMedDate not supplied

Tissue-specific protein kinase C isoform expression in rat uterine tissue.

OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a key role during the generation of agonist-stimulated phasic myometrial contractions. Protein kinase C (PKC), a component of this signaling pathway, has been previously shown to produce feedback inhibition of agonist-stimulated myometrial contractions. The studies described in this report were performed to survey the tissue-specific expression of several PKC isoforms in the rat uterus. METHODS: Uterine tissue was obtained from timed pregnant and normally cycling adult female Sprague-Dawley rats. Immunohistochemical studies were performed using the Vectastain ABC immunostaining technique and PKC isoform-specific polyclonal antibodies. Western blot studies were performed using myometrial tissue separated into cytosol and membrane fractions by differential centrifugation. RESULTS: These studies confirmed significant expression of the PKC-alpha, -beta 2, -delta, -eta, and -zeta isoforms in myometrium from pregnant and estrus rats, whereas only trace or no expression of the PKC-beta 1, -gamma, -epsilon, and -theta isoforms was observed. Expression of the PKC-alpha, -beta 2, and -eta isoforms decreased modestly during the latter days of gestation; in contrast, PKC-delta and -zeta remained stable during this period. The immunohistochemical studies confirmed expression of the PKC-alpha, -beta 2, -delta, -eta, and -zeta isoforms in both circular and longitudinal smooth-muscle layers of the near-term pregnant rat uterus. CONCLUSION: In summary, these studies have confirmed significant levels of expression of several isoforms of PKC in estrus and near-term pregnant rat uterine tissue, which was most prominent in the smooth-muscle cells of the myometrium.

Animals↗

PubMed2005

Gas6 induces Akt/mTOR-mediated mesangial hypertrophy in diabetic nephropathy.

BACKGROUND: We have already reported Gas6 is involved in glomerular hypertrophy observed in diabetic nephropathy. However, the molecular mechanisms involved in glomerular hypertrophy are still unknown, especially in vivo. METHODS: In vivo, diabetes was induced in rats and mice by streptozotocin (STZ) and the activation of the Akt/mTOR pathway in glomeruli was examined. In vitro, mesangial hypertrophy was assessed by [(3)H]leucine incorporation and measuring cell areas. RESULTS: Akt, p70 S6 kinase, and 4E-BP-1 were induced and phosphorylated in rat glomerular lysates after 12 weeks of STZ injection when mesangial and glomerular hypertrophy was observed. We then examined the role of Gas6 by treating STZ-rats with warfarin, and found that warfarin treatment inhibited the phosphorylation of these molecules as well as the hypertrophy. We next examined whether high glucose stimulation can induce the expression of Gas6/Axl in mesangial cells. Stimulation of the cells with 25 mmol/L of glucose increased the expression of Gas6/Axl and mesangial cell size compared with that with 5.6 mmol/L of glucose. This hypertrophic effect was abolished in mesangial cells derived from Gas6 knockout mice. We also found that LY294002 and rapamycin blocked Gas6-induced activation of the Akt/mTOR pathway and mesangial hypertrophy. Furthermore, less phosphorylated Akt-positive or 4E-BP-1-positive areas were found in STZ-treated Gas6 knockout mice than in STZ-treated wild-type mice. CONCLUSION: Our study indicates that the Akt/mTOR pathway is a key signaling cascade in Gas6-mediated mesangial and glomerular hypertrophy and revealed a crucial role of Gas6/Axl and the Akt/mTOR pathway in the development of diabetic nephropathy.

Adaptor Proteins, Signal Transducing↗

PubMed1989

Selective inhibition of angiotensin-II-mediated aldosterone secretion by 5-hydroxyeicosatetraenoic acid.

We have previously demonstrated that the 12-lipoxygenase (12-LO) pathway plays a key role in angiotensin-II (AII)-dependent aldosterone production. In the present study we examined the role of the 5LO pathway on AII-induced aldosterone secretion in rat glomerulosa cells in vitro. The 5LO product 5-hydroxyeicosatetraenoic acid (5HETE) and its unstable precursor 5-hydroxyperoxyeicosatetraenoic acid did not significantly alter basal aldosterone secretion in concentrations from 10(-9)-10(-7) M. In contrast, 5HETE reduced peak AII-induced aldosterone production from 59.1 +/- 9.0 to 37.96 +/- 7.2 ng/10(6) cells (P less than 0.01). This was accompanied by inhibition of the AII-stimulated rise in 12HETE production (10(-9)M AII, 160 +/- 4% of control; 10(-9) M AII plus 10(-7) M 5HETE, 90 +/- 1% of control production). However, 5HETE had no effect on the aldosterone response to potassium or ACTH, secretagogues that cause no activation of the 12LO pathway. These results suggest that the 5LO product 5HETE can selectively modulate AII-dependent aldosterone secretion. Further, the selective inhibitory effect of 5HETE on the AII effect in rat glomerulosa cells may be exerted by blockade of arachidonate metabolism via the 12LO pathway. These results suggest that the 5LO pathway may negatively modulate AII action in the adrenal zona glomerulosa.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed2003

Mechanisms of regulating the Raf kinase family.

The MAP Kinase pathway is a key signalling mechanism that regulates many cellular functions such as cell growth, transformation and apoptosis. One of the essential components of this pathway is the serine/threonine kinase, Raf. Raf (MAPKK kinase, MAPKKK) relays the extracellular signal from the receptor/Ras complex to a cascade of cytosolic kinases by phosphorylating and activating MAPK/ERK kinase (MEK; MAPK kinase, MAPKK) that phosphorylates and activates extracellular signal regulated kinase (ERK; mitogen-activated protein kinase, MAPK), which phosphorylates various cytoplasmic and nuclear proteins. Regulation of both Ras and Raf is crucial in the proper maintenance of cell growth as oncogenic mutations in these genes lead to high transforming activity. Ras is mutated in 30% of all human cancers and B-Raf is mutated in 60% of malignant melanomas. The mechanisms that regulate the small GTPase Ras as well as the downstream kinases MEK and extracellular signal regulated kinase (ERK) are well understood. However, the regulation of Raf is complex and involves the integration of other signalling pathways as well as intramolecular interactions, phosphorylation, dephosphorylation and protein-protein interactions. From studies using mammalian isoforms of Raf, as well as C. elegans lin45-Raf, common patterns and unique differences of regulation have emerged. This review will summarize recent findings on the regulation of Raf kinase.

Animals↗

PubMed2003

BRAF mutations in non-Hodgkin's lymphoma.

Ras proteins control signalling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. BRAF, which encodes an RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers. The activating mutation of BRAF is known to play a role in tumour development. As there have been no data on the BRAF mutation in non-Hodgkin's lymphoma (NHL), we analysed the genomic DNAs from 164 NHLs by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) for the detection of somatic mutations of BRAF (exons 11 and 15). Overall, we detected BRAF mutations in four NHLs (2.4%). Whereas most BRAF mutations in human cancers involved V599 of BRAF, all of the four BRAF mutations in the NHLs involved other amino acids (one G468A, two G468R and one D593G). To our knowledge, this is the first report on BRAF mutation in NHL, and the data indicate that BRAF is occasionally mutated in NHL, and suggest that BRAF mutation may contribute to the tumour development in some NHLs.

Cell Transformation, Neoplastic↗

PubMed2003

BRAF and KRAS mutations in stomach cancer.

Ras proteins control signaling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. BRAF, which encodes a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers. The activating mutation of BRAF is known to play a role in tumor development. As there have been no data on the BRAF mutation in stomach cancer, we analysed the genomic DNAs from 319 stomach carcinomas for the detection of somatic mutations of BRAF. Overall, we detected BRAF mutations in seven stomach carcinomas (2.2%). Five of the seven BRAF mutations involved Val 599, the previously identified hotspot, but the substituted amino acid (V599 M) was different from the most common BRAF mutation (V599E). The remaining two mutations involved a conserved amino acid (D593G). One tumor had both BRAF and KRAS mutations. This is the first report on BRAF mutation in stomach cancer, and the data indicate that BRAF is occasionally mutated in stomach cancer, and suggest that alterations of RAS pathway both by RAS and BRAF mutations contribute to the pathogenesis of stomach cancer.

Amino Acid Sequence↗

PubMed2001

Pathways of angiotensin-(1-7) metabolism in the kidney.

Angiotensin-(1-7) [Ang-(1-7)], which can be formed directly from angiotensin I (Ang I) bypassing the requisite production of Ang II, is a bioactive component of the renin-angiotensin system that may oppose the actions of Ang II. In contrast to the generation of Ang II, angiotensin-converting enzyme (ACE) hydrolyses Ang-(1-7) to inactive fragments. ACE inhibitors substantially augment circulating levels of Ang-(1-7) and increase the peptide's half-life. Thus, this enzymatic pathway constitutes a key regulatory point in the vasculature to balance the actions of Ang II, Ang-(1-7) and bradykinin. In contrast, the renal pathways for the metabolism of Ang-(1-7) appear quite distinct. Characterization of this pathway may shed new light on the potential actions of the peptide in the kidney, as well as the mechanisms of novel vasoactive peptidase therapies. We summarize recent experimental and clinical studies that begin to reveal novel pathways in the formation and degradation of Ang-(1-7) in the kidney.

Angiotensin I↗

PubMed2003

Sorbitol dehydrogenase: a novel target for adjunctive protection of ischemic myocardium.

Sorbitol dehydrogenase (SDH) is a polyol pathway enzyme that catalyzes conversion of sorbitol to fructose. Recent studies have demonstrated that activation of aldose reductase, the first enzyme of the polyol pathway, is a key response to ischemia and that inhibition of aldose reductase reduces myocardial ischemic injury. In our efforts to understand the role of pathway in affecting metabolism under normoxic and ischemic conditions, as well as in ischemic injury in myocardium, we investigated the importance of SDH by use of a specific inhibitor (SDI), CP-470,711. SDH inhibition increased glucose oxidation, whereas palmitate oxidation remained unaffected. Global ischemia increased myocardial SDH activity by approximately 1.5 fold. The tissue lactate/pyruvate ratio, a measure of cytosolic NADH/NAD+, was reduced by SDH inhibition under both normoxic and ischemic conditions. ATP was higher in SDI hearts during ischemia and reperfusion. Creatine kinase release during reperfusion, a marker of myocardial ischemic injury, was markedly attenuated in SDH-inhibited hearts. These data indicate that myocardial SDH activation is a component of ischemic response and that interventions that inhibit SDH protect ischemic myocardium. Furthermore, these data identify SDH as a novel target for adjunctive cardioprotective interventions.

Animals↗

PubMed2000

A caspase-independent cell clearance program. The LEI/L-DNase II pathway.

The discovery of caspase-mitochondrial pathway counts as one of the most important discovery in apoptosis biochemistry. Today, however, we begin to recognize its limits. Inhibition of caspase does not prevent cell death in many mammalian models. Targeted disruption of caspases does not impair every type of apoptosis. Other pathways, caspase independent, are now described. Here we present one of these pathways. It is a serine-protease dependent pathway and its key event is the transformation of LEI (a serine protease inhibitor) into L-DNase II (an endonuclease). When using this apoptotic pathway the cell activates, at the same time, its endonuclease activity (L-DNase II appears) and its protease activity (there is a release of inhibition of proteases).

Animals↗

PubMed2006

Recent advances in the research and development of RAF kinase inhibitors.

The RAS-RAF-MEK-ERK signaling pathway (ERK pathway) plays a key role in tumorigenesis and cancer progression. Mutations of RAS or B-RAF lead to a constitutive activation of the ERK pathway, which ultimately results in increased cell division, and cell survival. This review article focuses on the recent literature related to ERK pathway inhibitors, with a particular emphasis on RAF kinase inhibitors. Preclinical and clinical data for the RAF kinase inhibitor sorafenib (BAY 43-9006 tosylate), that was recently approved in the US for the treatment of advanced renal cell carcinoma, are also outlined.

Animals↗

PubMed1983

Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites.

Surveys of the antigenic properties of a wide range of variants of the H3N2 (Hong Kong) influenza virus subtype have revealed complex patterns of variants cocirculating during each of the main epidemic eras of the subtype. We determined hemagglutinin (HA) gene sequences for 14 isolates chosen to give the wildest possible spread of variant types. The addition of these data to existing HA gene sequence information for other variants provides a comprehensive picture of HA gene evolution during antigenic drift among H3N2 subtype viruses. The data reveal the existence of multiple evolutionary pathways during at least one period of development of the subtype and strikingly demonstrate that amino acid changes are limited to a small number of locations on the HA molecule during antigenic drift. The occurrence of sequential amino acid changes at key positions within these variable regions suggests that the HA structure has remained constant during subtype evolution so that only limited possibilities remain for further antigenic drift among H3N2 viruses.

Amino Acid Sequence↗

PubMed2005

Stem cell division is regulated by the microRNA pathway.

One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

Animals↗