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At least 937 records · Page 52Linked to original sources
PubMed2006

Genetic analysis of a novel pathway for D-xylose metabolism in Caulobacter crescentus.

Genetic data suggest that the oligotrophic freshwater bacterium Caulobacter crescentus metabolizes D-xylose through a pathway yielding alpha-ketoglutarate, comparable to the recently described L-arabinose degradation pathway of Azospirillum brasilense. Enzymes of the C. crescentus pathway, including an NAD(+)-dependent xylose dehydrogenase, are encoded in the xylose-inducible xylXABCD operon (CC0823-CC0819).

Arabinose↗
PubMed2006

Structural characterization of a specific glycopeptidolipid containing a novel N-acyl-deoxy sugar from mycobacterium intracellulare serotype 7 and genetic analysis of its glycosylation pathway.

The nontuberculous Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is distributed ubiquitously in the environment and is an important cause of respiratory and lymphatic disease in humans and animals. These species produce polar glycopeptidolipids (GPLs), and of particular interest is their serotype-specific antigenicity. Structurally, GPLs contain an N-acylated tetrapeptide-amino alcohol core that is glycosylated at the C terminal with 3,4-di-O-methyl rhamnose and at the d-allo-threonine with a 6-deoxy-talose. This serotype nonspecific GPL is found in all MAC species. The serotype-specific GPLs are further glycosylated with a variable haptenic oligosaccharide at 6-deoxy-talose. At present, 31 distinct serotype-specific GPLs have been identified on the basis of oligosaccharide composition, and the complete structures of 14 serotype-specific GPLs have been defined. It is considered that the modification of the GPL structure plays an important role in bacterial physiology, pathogenesis, and host immune responses. In this study, we defined the complete structure of a novel serotype 7 GPL that has a unique terminal amido sugar. The main molecular mass is 1,874, and attached to the tetrapeptide-amino alcohol core is the serotype 7-specific oligosaccharide unit of 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-2-O-methyl-beta-hexose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->2)-alpha-l-6-deoxy-talose. Moreover, we isolated and characterized the serotype 7-specific gene cluster involved in glycosylation of the oligosaccharide. Nine open reading frames (ORFs) were observed in the cluster. Based on the sequence homology, the ORFs are thought to participate in the biosynthesis of the serotype 7 GPL.

Carbohydrate Sequence↗
PubMed1981

Genetic analysis of the RecE pathway of genetic recombination in Escherichia coli K-12.

The RecE pathway of genetic recombination in Escherichia coli K-12 was defined to be the pathway that is utilized in deoxyribonucleic acid exonuclease V (ExoV)-defective cells which express constitutively recE+, the structural gene for deoxyribonucleic acid exonuclease VIII. Dependence on ExoVIII was shown by the occurrence in a recB21 sbcA23 strain of recombination deficiency mutations in recE, the structural gene for ExoVIII. Point mutations in recE were found as well as deletion mutations in which the entire Rac prophage, carrying recE, was lost. In addition, strain construction and mutagenesis revealed the dependence of the RecE pathway on recA+ and on recF+. Dependence on a fourth gene was shown by a mutation (rec-77) which does not map near the other genes. The problem of distinguishing the RecE pathway from that previously called RecF is discussed.

Escherichia coli↗
PubMed2002

Domain interactions on the ntr signal transduction pathway: two-hybrid analysis of mutant and truncated derivatives of histidine kinase NtrB.

We have used the yeast two-hybrid system to analyze protein-protein interactions mediated by domains of regulatory proteins of the ntr signal transduction system, including interactions among NtrB derivatives and their interactions with NtrC and PII from Klebsiella pneumoniae. Interactions took place only between proteins or protein domains belonging to the ntr signal transduction system and not between proteins or domains from noncognate regulators. NtrB and its transmitter domain, but not NtrC, CheA, or the cytoplasmic C terminus of EnvZ, interacted with PII. In addition, interaction of NtrB with NtrC, but not with PII, depended on the histidine phosphotransfer domain. Point mutation A129T, diminishing the NtrC phosphatase activity of NtrB, affected the strength of the signals between NtrC and the transmitter module of NtrB but had no impact on PII signals, suggesting that A129T prevents the conformational change needed by NtrB to function as a phosphatase for NtrC, rather than disturbing binding to PII.

Bacterial Proteins↗
PubMed1990

Selection and analysis of galactose metabolic pathway variants of a mouse liver cell line.

To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG). One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity. Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line. We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity. Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars. These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway. The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.

Animals↗
PubMed1999

Analysis of fast synaptic pathways in myenteric plexus of guinea pig ileum.

Most fast excitatory postsynaptic potentials (fEPSPs) recorded from guinea pig ileum myenteric plexus are mediated by acetylcholine acting at nicotinic receptors and ATP acting at P2X receptors. These studies examine length and polarity of projection of neurons releasing mediators of fEPSPs. Under ketamine-xylazine anesthesia, animals were sham treated or myenteric pathways were interrupted. After severed axons degenerated, fEPSPs were recorded at the operated site using conventional, intracellular electrophysiological methods and were classified as nicotinic or mixed on the basis of sensitivity to hexamethonium. Cholinergic and noncholinergic fEPSPs were recorded from small, operated segments, suggesting that some neurons have projections between adjacent ganglia. The mean amplitudes of nicotinic and mixed fEPSPs were reduced after circumferential and descending pathways degenerated. The proportion of nicotinic vs. mixed fEPSPs recorded from tissues lacking descending projections was greater than that recorded from sham-treated tissues, suggesting that fibers releasing noncholinergic mediators project aborally. Descending projections communicate with neurons in ganglia at least three rows aboral to their origin. The data suggest that fast noncholinergic neurotransmission could contribute to hexamethonium-resistant descending inhibition during the peristaltic reflex.

Animals↗
PubMed1991

Retinal projections in the freshwater butterfly fish, Pantodon buchholzi (Osteoglossoidei). I. Cytoarchitectonic analysis and primary visual pathways.

The freshwater butterfly fish, Pantodon buchholzi, is a member of the most primitive radiation of teleosts. The retinofugal projections were studied in this fish with autoradiographic and horseradish peroxidase (HRP) methods, and the cytoarchitecture of the retinorecipient regions in the diencephalon and pretectum was analyzed with Bodian-, cresylecht-violet- and acetylcholinesterase-reacted sections. The rostral diencephalon of Pantodon contains a large retinorecipient nucleus, not previously identified in any other fish, i.e. nucleus rostrolateralis. Other nuclei that are described correspond to those previously recognized in other species. The majority of retinorecipient nuclei are positive for acetylcholinesterase, particularly those in the pretectum, as has been found in other species of teleosts. Most of the retinofugal fibers decussate in the optic chiasm. Some fibers project via the axial optic tract to preoptic nuclei and a region in the rostral hypothalamus. Fibers leave the medial optic tract to terminate in nucleus rostrolateralis and in dorsal and ventral thalamic nuclei, accessory optic and tubercular nuclei, periventricular and central pretectal nuclei, and sparsely in the deep tectal fascicle and terminal field. Dorsal optic tract fibers project to the dorsal accessory optic nucleus, superficial and central pretectal nuclei, and superficial and deep tectal layers. Ventral optic tract fibers project to the superficial pretectum, accessory optic nuclei, posterior tuberculum, nucleus corticalis in the central pretectum, and superficial tectal layer. Fibers that remain in the ipsilateral optic tract project to most of the targets reached by contralaterally projecting fibers. A few fibers in the contralateral medial optic tract redecussate via the posterior commissure to reach the ipsilateral periventricular pretectum. No labeled retinopetal cells caudal to the olfactory bulb were identified in any of the HRP cases.

Acetylcholinesterase↗
PubMed1989

Physiologic analysis of central motor pathways--simultaneous recording from multiple relaxed muscles.

The central motor pathways were studied with the technique of percutaneous electrical stimulation of the central nervous system in 40 normal volunteers. There were no significant differences between the right and left sides, or between males and females. A linear correlation between the cortical latency (Lcor) or the spinal latency (Lsp) and body height was observed, however, there was no correlation between the central conduction time (CCT) (Lcor-Lsp) and body height. Consequently, the CCT appears to provide a reliable estimate of the function of central motor pathways. We have established normal values for eight muscles at rest that would be required for localizing a very small intraspinal lesion.

Adult↗
PubMed1986

Tracheal permeability in rats exposed to ozone. An electron microscopic and autoradiographic analysis of the transport pathway.

Exposure of rats to ozone (O3), 0.8 ppm increases the tracheal permeability to 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) about twofold but to 125I-bovine serum albumin (125I-BSA) to a lesser extent. It is generally believed that exposure to air pollutants causes perturbation of tight junctions and formation of intercellular channels for the passage of molecules from airway lumen to blood. We now report that a second mechanism, vesicular transport, is operative in the transepithelial movement of molecules, that this mechanism is speeded in tracheas of O3-exposed rats, and that there is a concurrent delay in movement of BSA from connective tissue to capillaries after O3 exposure. Horseradish peroxidase (HRP) instilled in trachea was taken up by endocytic vesicles, which could be localized in apical as well as basal regions of ciliated and nonciliated cells. A count of HRP-positive vesicles and measurement of their surface area revealed an approximate twofold increase in O3-exposed rats over that in control animals breathing clean air; this paralleled a twofold increase in transport of 99mTc-DTPA from tracheal lumen to blood. An autophagocytic process induced in tracheal epithelial cells by O3 is proposed. Despite the difference in the size of HRP and BSA, the 2 molecules migrated through common pathways and were colocalized in the luminal membranes as well as in endocytic vesicles and intercellular spaces in double labeling experiments involving simultaneous detection of HRP by cytochemistry and 125I-BSA by autoradiography. This procedure proved particularly useful in detecting a dramatic accumulation of 125I-BSA autoradiographic grains in subepithelial connective tissue and HRP accumulation in intercellular spaces and at the basal membrane-connective tissue junction in O3-exposed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway.

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.

Biological Transport↗
PubMed2006

Chemical genetic analysis of signal transduction pathways.

Covalent modification of proteins by phosphorylation represents a major mechanism of signal transduction that contributes to many physiological and pathophysiological processes. The enzymes that regulate protein phosphorylation (e.g., protein kinases and phosphatases) are, therefore, potential targets for the rational design of small-molecule drugs for the treatment of many diseases. A critical aspect of drug design is the reliability of proof-of-principle studies that demonstrate the therapeutic potential of the drug target. Studies using genetically modified mouse models that lack expression of the drug target using gene ablation and RNAi-based technologies have been widely employed. Recently, an alternative strategy using a chemical genetic approach has been proposed. Here, the authors discuss the strengths and weaknesses of the chemical genetic approach for studies of target validation.

Drug Design↗
PubMed2001

Analysis of the signal pathways involved in the regulation of fatty acid synthase gene expression by insulin and somatotropin.

Our previous studies have shown that somatotropin (ST) antagonizes insulin stimulation of fatty acid synthase (FAS) enzyme activity and gene transcription in adipocytes. In the present study, inhibitors of insulin and ST signaling pathways were used to dissect the mechanisms by which these hormones regulate FAS gene expression in 3T3-F442A adipocytes. Treating 3T3-F442A adipocytes with 10 microM PD98059, an inhibitor of mitogen-activated protein (MAP) kinase, did not affect the induction of FAS mRNA by insulin. When cells were cultured with H-89 (10 microM), GF109203X (10 microM), or staurosporine (100 nM), inhibitors of protein kinase A, protein kinase C, and Janus kinase (JAK) 2, respectively, the inhibitory effect of ST on FAS mRNA levels was not altered. However, H-89 significantly decreased the stimulatory effect of insulin on FAS mRNA abundance. Moreover, treatment with okadaic acid (1 microM), a serine/threonine phosphatase inhibitor, abolished the induction of FAS mRNA by insulin. These results suggest that serine/threonine dephosphorylation and protein kinase A-dependent pathways are involved in the regulation of FAS gene expression by insulin, but MAP kinase is probably not involved. Furthermore, our data indicate that protein kinase A, protein kinase C, and JAK2 do not mediate the effect of ST on regulation of FAS mRNA abundance.

3T3 Cells↗
PubMedDate not supplied

Analysis of the endocytic pathway upon intracellular transport of IgG molecules through Fc receptors.

The uniformly distributed Fc receptors (FcRs) on the surface of many cell types are involved in a variety of immune reactions by non-specifically facilitating the entry of antigen-specific IgG molecules to the cell. Such reactions may be beneficial to the organism when foreign antigens are involved, or harmful in cases of self antigens and viruses. In order to avoid the IgG-mediated self antigen presentation or viral infection in autoimmunity and viral attack respectively, we attempt in this study to inhibit the intracellular transport of antibodies. This blockage, however, implies: efficacy of inhibition, inability of de novo exocytosis of the internalised antibody and finally maintenance of normal cell growth and morphology. We thus concentrate our interest on the endocytic pathway followed by a neutralising antibody in murine trophoblast cells where we try to inhibit antibody intracellular transport by various agents according to the criteria set above. In our model-system, IFN-gamma, upon induction of FcRs, facilitates endocytosis of the anti-p21ras antibody which blocks in turn the IFN-gamma-induced surface class II major histocompatibility complex (MHC) expression. Using various intracellular transport inhibitors, we study the required conditions by which these compounds cancel the inhibitory action of anti-p21ras and allow induction of class II MHC molecules by IFN-gamma. The effectiveness of the inhibitors in a ranking order is shown as following: monodansyl cadaverine > didansyl cadaverine > pepstatin A > leupeptin > NH4Cl > brefeldin A > ZPCK > TPCK. From these inhibitors, only brefeldin A, leupeptin, pepstatin and ZPCK do not allow exocytosis of the antibody in the culture medium and only didansyl cadaverine, pepstatin and leupeptin maintain cell viability and morphology. However, by sequential elimination based on this study's established criteria, only pepstatin A and leupeptin are shown to be effective inhibitors to specific antibody intracellular transport, protecting also the cell's viability and physiology.

Animals↗
PubMed2002

Pharmacological analysis of signal transduction pathways required for mycobacterium tuberculosis-induced IL-8 and MCP-1 production in human peripheral monocytes.

Signalling cascades involved in chemokine production by human phagocytes following infection with Mycobacterium tuberculosis are still not defined. We used specific pharmacologic inhibitors to identify the signalling molecules which lead to interleukin (IL)-8 and MCP-1 production in human monocytes in response to M. tuberculosis infection. Inhibition of extracellular signal-regulated (ERK) or p38 mitogen-activated protein kinase by PD98059 and SB203580 respectively, significantly affected chemokine production. However, only the presence of both inhibitors completely blocked the release. A down-regulation of chemokine secretion was found in presence of inhibitors of protein kinase (PK)C and phospholipase C. Moreover, production depended on transcription activation via the nuclear factor-kappa B (NF-kappaB), as demonstrated by treatment with actinomycin D and caffeic acid phenethyl ester. In addition, activation of PKA and the phosphoinoside 3-kinase (PI-3k)/p70 ribosomal S6 kinase cascade was required to have maximal MCP-1 but not IL-8 production. In conclusion, this study provides evidence that multiple signal transduction pathways are involved in M. tuberculosis -induced chemokine secretion by human monocytes. Moreover, for the first time this report indicates that inhibitors of some signalling molecules are able to dissociate IL-8 from MCP-1 secretion. Differences in the regulatory pathways of chemokine production can potentially be exploited therapeutically.

Chemokine CCL2↗
PubMed2003

[Analysis of signal transduction pathways in cerebellar long-term depression by use of kinetic simulations].

It is essential to analyze molecular basis of synaptic plasticity systematically for understanding our psychological processes and psychological disorders. Recent progress identified many signaling molecules responsible for the regulation of synaptic plasticity. However, because multiple molecular signal transduction molecules and pathways are involved, the systematic relationship between synaptic plasticity and the currently known signal transduction pathways remains obscure. To address this issue, we here focused on cerebellar long-term depression (LTD), one of the well known synaptic plasticity. We took advantage of a simulation model for cerebellar LTD based on biochemical reactions, kinetics, and explored the behaviors of complex signal transduction pathways involved in cerebellar LTD.

Cerebellar Cortex↗