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At least 1,333 records · Page 74Linked to original sources
PubMed1998

Significance of the reductase-dependent pathway for the beta-oxidation of unsaturated fatty acids with odd-numbered double bonds. Mitochondrial metabolism of 2-trans-5-cis-octadienoyl-CoA.

The beta-oxidation of unsaturated fatty acids with odd-numbered double bonds proceeds by reduction of the double bond (reductase-dependent pathway) in addition to the well established isomerization of the double bond (isomerase-dependent pathway). The metabolic significance of the reductase-dependent pathway was assessed with 2-trans-5-cis-octadienoyl-CoA (2,5-octadienoyl-CoA) and its products, all of which are metabolites of alpha-linolenic acid. A kinetic evaluation of beta-oxidation enzymes revealed that the presence of a 5-cis double bond in the substrate most adversely affected the activity of 3-ketoacyl-CoA thiolase although not enough to become rate-limiting. Concentration-dependent and time-dependent measurements indicated that most (80%) of 2,5-octadienoyl-CoA is metabolized via the isomerase-dependent pathway. The reason for the greater flux through the isomerase-dependent pathway is the higher activity of L-3-hydroxyacyl-CoA dehydrogenase as compared with Delta3,Delta2-enoyl-CoA isomerase. These two enzymes catalyze the rate-limiting steps in the isomerase-dependent and reductase-dependent pathways, respectively. Once 2,5-octadienoyl-CoA is converted to 3,5-octadienoyl-CoA (perhaps fortuitously because of the presence of Delta3,Delta2-enoyl-CoA isomerase), the only effective route for its degradation is via the reductase-dependent pathway. It is concluded that the reductase-dependent pathway assures the degradation of 3,5-dienoyl-CoA intermediates, thereby preventing the depletion of free coenzyme A and a likely impairment of mitochondrial oxidative function.

Acyl Coenzyme A↗
PubMed1998

Electron partitioning between the two branching quinol-oxidizing pathways in Acanthamoeba castellanii mitochondria during steady-state state 3 respiration.

Amoeba mitochondria possess a respiratory chain with two quinol-oxidizing pathways: the cytochrome pathway and the cyanide-resistant alternative oxidase pathway. The ADP/O method, based on the non-phosphorylating property of alternative oxidase, was used to determine contributions of both pathways in overall state 3 respiration in the presence of GMP (an activator of the alternative oxidase in amoeba) and succinate as oxidizable substrate. This method involves pair measurements of ADP/O ratios plus and minus benzohydroxamate (an inhibitor of the alternative oxidase). The requirements of the method are listed and verified. When overall state 3 respiration was decreased by increasing concentrations of n-butyl malonate (a non-penetrating inhibitor of succinate uptake), the quinone reduction level declined. At the same time, the alternative pathway contribution decreased sharply and became negligible when quinone redox state was lower than 50%, whereas the cytochrome pathway contribution first increased and then passed through a maximum at a quinone redox state of 58% and sharply decreased at a lower level of quinone reduction. This study is the first attempt to examine the steady-state kinetics of the two quinol-oxidizing pathways when both are active and to describe electron partitioning between them when the steady-state rate of the quinone-reducing pathway is varied.

Acanthamoeba↗
PubMed1999

The MEK pathway is required for stimulation of p21(WAF1/CIP1) by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta)can induce the cyclin-dependent kinase inhibitors p21 and p15 in a variety of cell types. We have shown previously that Smad3 is required for the growth inhibitory activity of TGF-beta, whereas overexpression of Smads is not sufficient to activate the expression of p21 in HaCaT cells. These data suggest that an additional signaling pathway may be involved in stimulating p21 in HaCaT cells. Given the recent finding that the mitogen-activated protein kinase (MAPK) pathway can cause p21 induction and arrest cells, we examined the involvement of this pathway for p21 and p15 induction by TGF-beta. We found that TGF-beta can regulate the MAPK pathway, leading to the increased transactivation ability of transcription factor Elk. Constitutively active components in the MAPK pathway activate p21 expression, and inhibitors or dominant negative constructs for the MAPK pathway significantly decrease p21 induction by TGF-beta. Both constitutively active MEK and inhibitors for MEK have no effect on Smad activity, including DNA binding, localization, and interaction with coactivator p300/CBP. These findings suggest that the MAPK pathway may be an independent pathway that is involved in p21 and p15 induction by TGF-beta.

Benzoquinones↗
PubMed2000

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation.

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.

Androstadienes↗
PubMed2001

Hepatitis B virus X protein differentially activates RAS-RAF-MAPK and JNK pathways in X-transforming versus non-transforming AML12 hepatocytes.

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronic HBV patients by an unknown mechanism. Activities of pX likely relevant to hepatocyte transformation include activation of the mitogenic RAS-RAF-MAPK and JNK pathways. To assess the importance of mitogenic pathway activation by pX in transformation, we employed a cellular model system composed of two tetracycline-regulated, pX-expressing cell lines, constructed in AML12-immortalized hepatocytes. This system includes the differentiated 3pX-1 and the de-differentiated 4pX-1 hepatocytes. Our studies have demonstrated that conditional pX expression transforms only 3pX-1 cells. Here, comparative in vitro kinase assays and various in vivo analyses demonstrate that pX affects an inverse activation of RAS-RAF-MAPK and JNK pathways in 3pX-1 versus 4pX-1 cells. Sustained pX-dependent RAS-RAF-MAPK pathway activation is observed in pX-transforming 3pX-1 cells, whereas sustained pX-dependent JNK pathway activation is observed in pX non-transforming 4pX-1 cells. This differential, pX-dependent mitogenic pathway activation affects differential activation of cAMP-response element-binding protein and c-Jun and determines the proliferative response of 3pX-1 and 4pX-1 cells. Furthermore, tetracycline-regulated, pX-NLS-expressing cell lines demonstrate that expression of the nuclear pX-NLS variant minimally activates the RAS-RAF-MAPK pathway and results in markedly reduced transformation. These results link sustained, pX-mediated activation of RAS-RAF-MAPK pathway to hepatocyte transformation.

Cell Division↗
PubMed2001

Telomerase can inhibit the recombination-based pathway of telomere maintenance in human cells.

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.

Chromosome Aberrations↗
PubMed2002

Interaction of the c-Jun/JNK pathway and cyclin-dependent kinases in death of embryonic cortical neurons evoked by DNA damage.

DNA damage, an important initiator of neuronal death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic cortical neuronal death evoked by the DNA-damaging agent, camptothecin. In this model, the tumor suppressor p53 and cyclin-dependent kinases (CDKs) are activated independently and cooperate to mediate the conserved death pathway. To further our understanding, we presently examined whether the c-Jun/JNK pathway modulates death and whether this pathway is regulated by CDKs, p53, and Bax. We show that c-Jun/JNK is activated following DNA damage. Moreover, the c-Jun pathway is one mediator of death, because expression of dominant negative c-Jun and cdc42, and JNK pathway inhibitors are neuroprotective. Although previous evidences indicate that JNK3 is required for neuronal death under certain conditions, we show that JNK3 deficiency only partially mediates c-Jun phosphorylation and its deficiency does not protect neurons from death. Interestingly, we provide evidence that CDK activity regulates c-Jun but does not affect upstream pathways that lead to JNK phosphorylation. Finally, c-Jun activation is independent of p53 and Bax. Accordingly, we propose that c-Jun is regulated by the JNK and CDK pathways and that both must be activated for efficient c-Jun activation to occur.

Animals↗
PubMed2002

Cooperative binding of the cytoplasm to vacuole targeting pathway proteins, Cvt13 and Cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy.

Autophagy is a catabolic membrane-trafficking mechanism involved in cell maintenance and development. Most components of autophagy also function in the cytoplasm to vacuole targeting (Cvt) pathway, a constitutive biosynthetic pathway required for the transport of aminopeptidase I (Ape1). The protein components of autophagy and the Cvt pathway include a putative complex composed of Apg1 kinase and several interacting proteins that are specific for either the Cvt pathway or autophagy. A second required complex includes a phosphatidylinositol (PtdIns) 3-kinase and associated proteins that are involved in its activation and localization. The majority of proteins required for the Cvt and autophagy pathways localize to a perivacuolar pre-autophagosomal structure. We show that the Cvt13 and Cvt20 proteins are required for transport of precursor Ape1 through the Cvt pathway. Both proteins contain phox homology domains that bind PtdIns(3)P and are necessary for membrane localization to the pre-autophagosomal structure. Functional phox homology domains are required for Cvt pathway function. Cvt13 and Cvt20 interact with each other and with an autophagy-specific protein, Apg17, that interacts with Apg1 kinase. These results provide the first functional connection between the Apg1 and PtdIns 3-kinase complexes. The data suggest a role for PtdIns(3)P in the Cvt pathway and demonstrate that this lipid is required at the pre-autophagosomal structure.

Amino Acid Sequence↗
PubMed2003

Multiple, independently regulated pathways of cholesterol transport across the intestinal epithelial cells.

The present study provides a new understanding about the mechanisms involved in cholesterol absorption by the intestinal cells. Contrary to general belief, our data show that newly absorbed cholesterol is neither immediately available for secretion with apoB lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated, apoB-dependent and -independent, pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein and involve efflux mediated by ABCA1, high density lipoprotein assembly, and possibly other unknown mechanisms. There are at least two different metabolic pools of cholesterol. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. The esterified cholesterol is mainly secreted by the apoB-dependent pathway, whereas both the pathways are involved in the secretion of free cholesterol. Thus, enterocytes transport exogenous cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport.

Animals↗
PubMed2004

Impact of the mitogen-activated protein kinase pathway on parathyroid hormone-related protein actions in osteoblasts.

Parathyroid hormone-related protein (PTHrP) regulates proliferation and differentiation of osteoblastic cells via binding to the parathyroid hormone receptor (PTH-1R). The cAMP-dependent protein kinase A pathway governs the majority of these effects, but recent evidence also implicates the MAPK pathway. MC3T3-E1 subclone 4 cells (MC4) were treated with the MAPK inhibitor U0126 and PTHrP. In differentiated MC4 cells, osteocalcin and bone sialoprotein gene expression were both down-regulated by PTHrP and also by inhibition of the MAPK pathway. PTHrP-mediated down-regulation of PTH-1R mRNA and up-regulation of c-fos mRNA were MAPK-independent, whereas PTHrP stimulation of fra-2 and interleukin-6 (IL-6) mRNA was MAPK-dependent. Luciferase promoter assays revealed that regulation of IL-6 involved the cAMP-dependent protein kinase A and MAPK pathways with a potential minor role of the protein kinase C pathway, and a promoter region containing an activator protein-1 site was necessary for PTHrP-induced IL-6 gene transcription. An alternative pathway, through cAMP/Epac/Rap1/MAPK, mediated ERK phosphorylation but was not sufficient for IL-6 promoter activation. Phosphorylation of the transcription factor CREB was also necessary but not sufficient for PTHrP-mediated IL-6 promoter activity. Most interesting, a bidirectional effect was found with PTHrP increasing phosphorylated ERK in undifferentiated MC4 cells but decreasing phosphorylated ERK in differentiated cells. These data indicate that inactivation of the MAPK pathway shows differential regulation of PTHrP-stimulated activator protein-1 members, blocks PTHrP-stimulated IL-6, and synergistically down-regulates certain osteoblastic markers associated with differentiation. These novel findings indicate that the MAPK pathway plays a selective but important role in the actions of PTHrP.

Animals↗
PubMed2004

The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae.

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.

Autophagy↗
PubMed2004

Expression of the HXT1 low affinity glucose transporter requires the coordinated activities of the HOG and glucose signalling pathways.

Expression of the HXT1 gene, which encodes a low affinity glucose transporter in Saccharomyces cerevisiae, is regulated positively in response to glucose by the general glucose induction pathway, involving the Snf3/Rgt2 membrane glucose sensors, the SCF-Grr1 ubiquitination complex and the Rgt1 transcription factor. In this study we show that, in addition to the glucose signaling pathway, regulation of HXT1 expression also requires the HOG pathway. Deletion of components in the glucose signaling pathway or in the HOG pathway results in impaired HXT1 expression. Genetic analyses showed that, whereas the glucose signaling pathway regulates HXT1 through modulation of the Rgt1 transcription factor, the HOG pathway modulates HXT1 through regulation of the Sko1-Tup1-Ssn6 complex. Coordinated regulation of the two signaling pathways is required for expression of HXT1 by glucose and in response to osmostress.

Gene Deletion↗
PubMed2004

Basic calcium phosphate crystals activate p44/42 MAPK signal transduction pathway via protein kinase Cmicro in human fibroblasts.

Although basic calcium phosphate (BCP) crystals are common in osteoarthritis, the crystal-induced signal transduction pathways in human fibroblasts have not been fully comprehended. We have previously demonstrated that the induction of matrix metalloproteinases (MMP) 1 and 3 by BCP crystals follows both the calcium-dependent protein kinase C (PKC) pathway and the calcium-independent p44/42 mitogen-activated protein kinase (p44/42 MAPK) pathway. Although we showed that the calcium-dependent PKC pathway was characterized by calcium-dependent PKCalpha, here we show that the calcium-independent p44/42 MAPK pathway is mediated by calcium-independent PKCmicro. Inhibition of PKCmicro synthesis and activity by antisense oligodeoxynucleotides and H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide), respectively, results in the inhibition of p44/42 MAPK activation, thus demonstrating that p44/42 MAPK activity is dependent upon PKCmicro. Reverse transcription-polymerase chain reaction and Western blotting also show that inhibition of PKCmicro results in the inhibition of MMP-1 and MMP-3 mRNA and protein expression as a result of p44/42 MAPK inhibition. These results now lead us to the conclusion that BCP crystal activation of human fibroblasts follows two pathways: 1) the calcium-dependent PKC pathway characterized by PKCalpha and 2) the calcium-independent p44/42 MAPK pathway mediated by PKCmicro, which operate independently leading to an increase in mitogenesis and MMP synthesis and ultimately complementing each other for the efficient regulation of cellular responses to BCP crystal stimulation of human fibroblasts.

Calcium Phosphates↗
PubMed2005

Extracellular matrix-mediated signaling by dentin phosphophoryn involves activation of the Smad pathway independent of bone morphogenetic protein.

Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323-53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-beta family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development.

Active Transport, Cell Nucleus↗
PubMed2006

Up-regulation of the Wnt, estrogen receptor, insulin-like growth factor-I, and bone morphogenetic protein pathways in C57BL/6J osteoblasts as opposed to C3H/HeJ osteoblasts in part contributes to the differential anabolic response to fluid shear.

C57BL/6J (B6), but not C3H/HeJ (C3H), mice responded to mechanical loading with an increase in bone formation. A 30-min steady fluid shear of 20 dynes/cm(2) increased [(3)H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin beta1 (Igtb1) and cyclooxygenase-2 (Cox-2)) in B6 but not C3H osteoblasts, indicating that the differential mechanosensitivity was intrinsic to osteoblasts. In-house microarray analysis with 5,500 gene fragments revealed that the expression of 669 genes in B6 osteoblasts and 474 genes in C3H osteoblasts was altered 4 h after the fluid shear. Several genes associated with the insulin-like growth factor (IGF)-I, the estrogen receptor (ER), the bone morphogenetic protein (BMP)/transforming growth factor-beta, and Wnt pathways were differentially up-regulated in B6 osteoblasts. In vitro mechanical loading also led to up-regulation of these genes in the bones of B6 but not C3H mice. Pretreatment of B6 osteoblasts with inhibitors of the Wnt pathway (endostatin), the BMP pathway (Noggin), or the ER pathway (ICI182780) blocked the fluid shear-induced proliferation. Inhibition of integrin and Cox-2 activation by echistatin and indomethacin, respectively, each blocked the fluid shear-induced up-regulation of genes associated with these four pathways. In summary, up-regulation of the IGF-I, ER, BMP, and Wnt pathways is involved in mechanotransduction. These four pathways are downstream to the early mechanoresponsive genes, i.e. Igtb1 and Cox-2. In conclusion, differential up-regulation of these anabolic pathways may in part contribute to the good and poor response, respectively, in the B6 and C3H mice to mechanical loading.

Animals↗
PubMed2006

A functional transsulfuration pathway in the brain links to glutathione homeostasis.

Oxidative stress and diminished glutathione pools play critical roles in the pathogenesis of neurodegenerative diseases, including Alzheimer and Parkinson disease. Synthesis of glutathione, the most abundant mammalian antioxidant, is regulated at the substrate level by cysteine, which is synthesized from homocysteine via the transsulfuration pathway. Elevated homocysteine and diminished glutathione levels, seen in Alzheimer and Parkinson disease patients suggest impairments in the transsulfuration pathway that connects these metabolites. However, the very existence of this metabolic pathway in the brain is a subject of controversy. The product of the first of two enzymes in this pathway, cystathionine, is present at higher levels in brain as compared with other organs. This, together with the reported absence of the second enzyme, gamma-cystathionase, has led to the suggestion that the transsulfuration pathway is incomplete in the brain. In this study, we incubated mouse and human neurons and astrocytes and murine brain slices in medium with [35S]methionine and detected radiolabel incorporation into glutathione. This label transfer was sensitive to inhibition of gamma-cystathionase. In adult brain slices, approximately 40% of the glutathione was depleted within 10 h following gamma-cystathionase inhibition. In cultured human astrocytes, flux through the transsulfuration pathway increased under oxidative stress conditions, and blockade of this pathway led to reduced cell viability under oxidizing conditions. This study establishes the presence of an intact transsulfuration pathway and demonstrates its contribution to glutathione-dependent redox-buffering capacity under ex vivo conditions in brain cells and slices.

Animals↗
PubMed2006

Identification and characterization of L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase, and L-arabinolactonase involved in an alternative pathway of L-arabinose metabolism. Novel evolutionary insight into sugar metabolism.

Azospirillum brasiliense possesses an alternative pathway of L-arabinose metabolism, different from the known bacterial and fungal pathways. In the preceding articles, we identified and characterized L-arabinose-1-dehydrogenase and alpha-ketoglutaric semialdehyde dehydrogenase, which catalyzes the first and final reaction steps in this pathway, respectively (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623 and Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888). We here report the remaining three enzymes, L-arabonate dehydratase, L-2-keto-3-deoxyarabonate (L-KDA) dehydratase, and L-arabinolactonase. N-terminal amino acid sequences of L-arabonate dehydratase and L-KDA dehydratase purified from A. brasiliense cells corresponded to those of AraC and AraD genes, which form a single transcriptional unit together with the L-arabinose-1-dehydrogenase gene. Furthermore, the L-arabinolactonase gene (AraB) was also identified as a component of the gene cluster. Genetic characterization of the alternative L-arabinose pathway suggested a significant evolutional relationship with the known sugar metabolic pathways, including the Entner-Doudoroff (ED) pathway and the several modified versions. L-arabonate dehydratase belongs to the ILVD/EDD family and spectrophotometric and electron paramagnetic resonance analysis revealed it to contain a [4Fe-4S](2+) cluster. Site-directed mutagenesis identified three cysteine ligands essential for cluster coordination. L-KDA dehydratase was sequentially similar to DHDPS/NAL family proteins. D-2-Keto-3-deoxygluconate aldolase, a member of the DHDPS/NAL family, catalyzes the equivalent reaction to L-KDA aldolase involved in another alternative L-arabinose pathway, probably associating a unique evolutional event between the two alternative L-arabinose pathways by mutation(s) of a common ancestral enzyme. Site-directed mutagenesis revealed a unique catalytic amino acid residue in L-KDA dehydratase, which may be a candidate for such a natural mutation.

Amino Acid Sequence↗
PubMed2003

Dual AV nodal pathways and conduction during atrial fibrillation.

OBJECTIVE: AV node modification reduces ventricular rate during atrial fibrillation (AF). We induced AF in patients with dual AV nodal pathways before and after radiofrequency ablation (RFA) of AV nodal reentry tachycardia (AVNRT) and examined the role of the two pathways in the transmission of impulses during AF. DESIGN AND RESULTS: AF was induced in 30 patients before and after slow pathway ablation. Before RFA mean (AF CLmean) and shortest (AF CLshort) ventricular cycle lengths correlated significantly to ERPf, ERPs, and antegrade Wenckebach block (r = 0.53-0.67). Ablation eliminated slow pathway conduction completely in 10 patients (group A), whereas in 20 patients some slow pathway conduction was still present (group B). After RFA there was a 10% increase in AF CLmean (20%, p < 0.05 in A and 5%, p = NS in B) and 7% in AF CLshort (11%, p = NS in A and 6%, p = NS in B). During isoproterenol infusion after RFA AF CLmean increased 8% (p < 0.05) (14% in A and 6% in B; p < 0.05 in both groups). The effects of RFA were mainly confined to patients with ERPs less than the median value (13% vs 3% in those above median, respectively; p < 0.05). CONCLUSION: The refractory periods of the AV nodal pathways are the main determinants of ventricular rate during induced AF. Slow pathway ablation reduces ventricular rate during AF. This effect was greatest when slow pathway conduction was completely eliminated. A short ERPs predicted a greater reduction in ventricular rate.

Adrenergic beta-Agonists↗