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PubMed1998

Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Proteomic analysis of the brain in Alzheimer's disease: molecular phenotype of a complex disease process.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder accounting for about 50% of all dementias, yet its pathogenic mechanisms remain poorly understood. In order to provide a more complete picture of pathogenesis in AD, we analysed six human brain regions for alterations in their proteomes. Quantitative proteome analysis was used to compare signals corresponding to individual proteins between post mortem brain tissues from persons with AD, and those from age-matched nondemented control (NC) tissues. In severely injured brain regions, 76 proteins were differentially expressed in AD hippocampus compared with NC, 62 proteins were differentially expressed in temporal cortex, and 39 proteins were differentially expressed in entorhinal cortex. Significant differences were also present in relatively spared regions. Thus, 34 proteins were differentially expressed in AD cerebellum compared with NC, 125 proteins were differentially expressed in cingulate gyrus, and 75 proteins were differentially expressed in sensorimotor cortex. The identity of 37 of these proteins was determined, and the possible relevance of changes in key pathogenic pathways analysed. These studies provide a unique snapshot illustrating the complexity of interrelated disease mechanisms at work in a complex, multifactorial disease, and show that comparative proteome analysis is a method with the power to develop important new insights into pathogenic mechanisms in the dementias.

Alzheimer Disease↗

PubMed2003

Tumor necrosis factor alpha activation of the apoptotic cascade in murine articular chondrocytes is associated with the induction of metalloproteinases and specific pro-resorptive factors.

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) blockade provides substantive reduction of the symptoms of rheumatoid arthritis (RA). While the biologic actions of TNFalpha have been well characterized in immune and synovial cells, which are known to be major contributors to the progression of cartilage destruction in RA, the current studies were designed to assess the direct effects of TNFalpha on chondrocytes. METHODS: We examined the expression of several groupings of messenger RNA (mRNA) that define key biologic pathways that have previously been associated with either the general actions of TNFalpha or cartilage destruction, in murine articular chondrocytes isolated from wild-type mice and TNFalpha receptor-null (p55/p75(-/-)) mice. RESULTS: TNFalpha induced the expression of multiple mRNA that facilitate apoptosis and lead to apoptosis-induced cell death. The induction of apoptosis was accompanied by the increased expression of several factors involved in the regulation of skeletal tissue proteolysis and resorption. Quantitative increases from 2-fold to >10-fold were seen for inducible nitric oxide synthase, matrix metalloproteinase 3, macrophage colony-stimulating factor, and osteoprotegerin mRNA expression. The dependence of the induction of these mRNA on TNFalpha was confirmed by comparison with the effects of TNFalpha on chondrocytes isolated from receptor-null mice. CONCLUSION: These findings demonstrate that TNFalpha alters the expression of a complex array of genes within murine chondrocytes that contribute to the destruction of joint surfaces, independent of its actions on synovial and immune cells. Further studies are needed to clarify the biologic actions of TNFalpha in human cartilage cells.

Animals↗

PubMed2005

Inhibition of inflammatory bone erosion by constitutively active STAT-6 through blockade of JNK and NF-kappaB activation.

OBJECTIVE: NF-kappaB and JNK signaling pathways play key roles in the pathogenesis of inflammatory arthritis. Both factors are also activated in response to osteoclastogenic factors, such as RANKL and tumor necrosis factor alpha. Inflammatory arthritis and bone erosion subside in the presence of antiinflammatory cytokines such as interleukin-4 (IL-4). We have previously shown that IL-4 inhibits osteoclastogenesis in vitro through inhibition of NF-kappaB and JNK activation in a STAT-6-dependent manner. This study was undertaken to investigate the potential of constitutively active STAT-6 to arrest the activation of NF-kappaB and JNK and to subsequently ameliorate the bone erosion associated with inflammatory arthritis in mice. METHODS: Inflammatory arthritis was induced in wild-type and STAT-6-null mice by intraperitoneal injection of arthritis-eliciting serum derived from K/BxN mice. Bone erosion was assessed in the joints by histologic and immunostaining techniques. Cell-permeable Tat-STAT-6 fusion proteins were administered intraperitoneally. Cells were isolated from bone marrow and from joints for the JNK assay, the DNA-binding assays (electrophoretic mobility shift assays), and for in vitro osteoclastogenesis. RESULTS: Activation of NF-kappaB and JNK in vivo was increased in extracts of cells retrieved from the joints of arthritic mice. Cell-permeable, constitutively active STAT-6 (i.e., STAT-6-VT) was effective in blocking NF-kappaB and JNK activation in RANKL-treated osteoclast progenitors. More importantly, STAT-6-VT protein significantly inhibited the in vivo activation of NF-kappaB and JNK, attenuated osteoclast recruitment in the inflamed joints, and decreased bone destruction. CONCLUSION: Our findings indicate that the administration of STAT-6-VT presents a novel approach to the alleviation of bone erosion in inflammatory arthritis.

Animals↗

PubMed2004

Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures.

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.

Cell Culture Techniques↗

PubMed2006

Understanding sulfone behavior in palladium-catalyzed domino reactions with aryl iodides.

Unlike traditionally used acyclic 1,2-disubstituted alkenes, the reaction of alpha,beta-unsaturated phenyl sulfones with aryl iodides under Heck reaction conditions takes place mainly by means of a four-component domino process, involving one unit of the alkene and three units of the aryl iodide, affording substituted 9-phenylsulfonyl-9,10-dihydrophenanthrenes. We report here the results of a computational study on the mechanism of this domino arylation reaction. Based on these results we can explain why vinyl sulfones, unlike other electron-deficient alkenes such as enones, preferentially follow this domino pathway instead of the usual Heck pathway. The key step is a C-H activation process in which a five-membered palladacycle is formed. The greater ability of vinyl sulfones, relative to enones, to reach the transition state that leads to the formation of the initial palladacycle makes the difference.

Journal Article↗

PubMed2006

Shh/BMP-4 signaling pathway is essential for intestinal epithelial development during Xenopus larval-to-adult remodeling.

During amphibian larval-to-adult intestinal remodeling, progenitor cells of the adult epithelium actively proliferate and differentiate under the control of thyroid hormone (TH) to form the intestinal absorptive epithelium, which is analogous to the mammalian counterpart. We previously found that TH-up-regulated expression of bone morphogenetic protein-4 (BMP-4) spatiotemporally correlates with adult epithelial development in the Xenopus laevis intestine. Here, we aimed to clarify the role of BMP-4 in intestinal remodeling. Our reverse transcriptase-polymerase chain reaction and in situ hybridization analyses indicated that mRNA of BMPR-IA, a type I receptor of BMP-4, is expressed in both the developing connective tissue and progenitor cells of the adult epithelium. More importantly, using organ culture and immunohistochemical procedures, we have shown that BMP-4 not only represses cell proliferation of the connective tissue but promotes differentiation of the intestinal absorptive epithelium. In addition, we found that the connective tissue-specific expression of BMP-4 mRNA is up-regulated by sonic hedgehog (Shh), whose epithelium-specific expression is directly induced by TH. These results strongly suggest that the Shh/BMP-4 signaling pathway plays key roles in the amphibian intestinal remodeling through epithelial-connective tissue interactions.

Animals↗

PubMed2005

Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood.

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.

CD11b Antigen↗

PubMed2004

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen-sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR).

We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with androgens further reduced invasion of the cells without modifying alpha6beta4 expression, suggesting an interference with the invasion process by androgens. Here, we investigated EGF-mediated signal transduction processes that lead to invasion in PC3-AR cells. We show that EGF-induced EGFR autotransphosphorylation is reduced in PC3-AR cells compared to PC3 cells transfected only with the vector (PC3-Neo). EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, and EGF-PI3K interaction are also decreased in PC3-AR cells and further reduced by treatment with androgen. Finally, we show that EGFR internalization process was reduced in PC3-AR and LNCaP cells compared to PC3-Neo. Investigations on the location of AR in PC3-AR transfected cells were also conducted. Immunoconfocal microscopy and coimminoprecipitation studies demonstrated the presence of an interaction between EGFR and AR at membrane level in PC3-AR and LNCaP cells. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less-malignant phenotype by interfering with EGFR signaling leading to invasion through a mechanism involving an interaction between AR and EGFR.

Androgens↗

PubMed1995

(Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima.

To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase.

Aldose-Ketose Isomerases↗

PubMed2006

The effect of hydroxyapatite nanocrystals on microvascular endothelial cell viability and functions.

To favor bone reconstitution with biomaterials endothelial cells should maintain proper functions to drive angiogenesis. To this aim nanocrystals of hydroxyapatite (HA) have been synthesized and characterized on endothelial cells. Microvascular endothelial cells have been exposed to stoichiometric HA nanocrystals. Cell morphology and organization of cytoskeletal proteins have been monitored by SEM analysis and immunofluorescence. Biochemical markers of physiological and pathological responses of endothelial cells, endothelial constitutive nitric oxide synthase, and cycloxygenase-2 (ecNOS and COX-2, respectively) have been measured by immunofluorescence. Crystallized HA sustained endothelial survival without any cytotoxic effect. At the observation with SEM, endothelial cell morphology was maintained in the presence of HA. The localization and organization of beta-actin documented the formation of stress fibers, indicating an activation of endothelial cells induced by HA nanocrystals. Immunohistochemistry for biochemical key signaling pathways in endothelium demonstrated that nanocrystals of HA maintained the expression of ecNOS and did not increase COX-2 expression. In conclusion, the present findings indicate that HA nanocrystals exhibit high biocompatibility for microvascular endothelium. In the presence of HA nanocrystals endothelial cells maintain biochemical markers of healthy endothelium. They do not acquire a proinflammatory or thrombogenic phenotype, but express markers of functioning endothelium that might contribute to angiogenesis.

Actins↗

PubMed2006

Possible role of duration of PKC-induced ERK activation in the effects of agonists and phorbol esters on DNA synthesis in Panc-1 cells.

Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) have been implicated in the effects of regulatory peptides on proliferation. We studied how ERK was activated by PKC following regulatory peptide or phorbol ester stimulation and we also investigated the effect of ERK activation on proliferation in Panc-1 cells. Panc-1 cells transfected with CCK1 receptors were treated with cholecystokinin (CCK), neurotensin (NT), or phorbol 12-myristate 13-acetate (PMA). DNA synthesis was studied by measuring tritiated thymidine incorporation. PKC isoforms were selectively inhibited with Gö6983 and 200 nM Ro-32-0432, their translocation was detected by confocal microscopy and by subcellular fractionation followed by immunoblotting. ERK cascade activation was detected with phosphoERK immunoblotting and inhibited with 20 microM PD98059. PMA and CCK inhibited, NT stimulated DNA synthesis. These effects were inhibited by Ro-32-0432 but not by Gö6983 suggesting the involvement of PKCepsilon in proliferation control. Confocal microscopy and subcellular fractionation demonstrated that PMA, CCK, and NT caused cytosol to membrane translocation of PKCepsilon and ERK activation that was inhibited by Ro-32-0432 but not by Gö6983. ERK activation was prolonged following PMA and CCK, but transient after NT treatment. PMA, CCK, and NT all activated cyclinD1, while p21CIP1 expression was increased by only PMA and CCK, but not by NT; each of these effects is inhibited by PD98059. In conclusion, our results provide evidence for PKCepsilon-mediated differential ERK activation and growth regulation in Panc-1C cells. Identification of the mechanisms by which these key signaling pathways are modulated could provide a basis for the development of novel therapeutic interventions to treat pancreatic cancer.

Cell Line, Tumor↗

PubMed2003

Relative contributions of neural mechanisms versus muscle mechanics in promoting finger extension deficits following stroke.

The origins of impaired finger and hand function were examined in 10 stroke survivors with chronic spastic hemiparesis, with the intent of assessing whether mechanical restraint or altered neurophysiological control mechanisms are responsible for the well-known impairment of finger extension. Simultaneous extension of all four metacarpophalangeal (MCP) joints of the impaired hand was either externally imposed using a rotary actuator or attempted voluntarily by the subject. Trials were conducted both before and after administration of a local anesthetic, blocking the median and ulnar nerves at the elbow. The anesthetic was administered to reduce the activity of the muscles flexing the MCP joints, in order to distinguish mechanical from neuronal resistance to imposed MCP rotation. We found that the nerve blockade resulted in a reduction in velocity-dependent torque (P = 0.01), thereby indicating significant joint impedance due to spasticity. Blockade also produced a posture-dependent reduction in static torque in declaratively relaxed subjects (P = 0.04), suggesting some tonic flexor activity for specific hand postures. No change in either extensor isometric (P = 0.33) or isokinetic (0.53) torque was apparent, but 3 of the 10 subjects did exhibit substantial (>10 degrees ) improvement in voluntary MCP extension following the blockade. This improvement seemed largely due to a decrease in inappropriate flexor activity during the movement, rather than an increase in extensor activity. We argue that persistent and inappropriate flexor activation plays a role in limiting voluntary finger extension, and that this activation is potentially a reflection of altered supraspinal control of key spinal pathways. In all cases, this inappropriate activation was compounded by weakness, apparent in both the extensor and flexor muscles.

Biomechanical Phenomena↗

PubMedDate not supplied

Direct analysis of abscisic acid in crude plant extracts by liquid chromatography--electrospray/tandem mass spectrometry.

A new method, based on liquid chromatography--electrospray/tandem mass spectrometry, for the determination of abscisic acid (ABA), an essential plant hormone that regulates key metabolic pathways and responses to environmental cues, has been developed. Substantial changes in extraction procedures are also proposed. Data indicate that the organic solvents classically used as extraction buffers can be substituted by an aqueous solution, resulting in the same amounts of extracted ABA. The new method, which uses minute amounts of plant tissue, has an estimated limit of detection below 50 pmol ABA/g, and the sensitivity of the technique allows the analysis of ABA in crude plant extracts. Overall, this new rapid, sensitive and accurate procedure to determine ABA will allow analysis of multiple samples in a short time and represents a clear advantage in comparison with the conventional procedures involving many preparative steps and large amounts of plant tissue.

Abscisic Acid↗

PubMed2007

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica.

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.

Amino Acid Sequence↗

PubMed1999

The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates.

A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.

Acetylcysteine↗

PubMed2000

Amino acid uptake and regulation in multicellular hepatoma spheroids.

BACKGROUND: Cancer cells maintained in monolayer tissue culture are frequently used to study tumor biology and nutrient uptake, but there is a concern that this system may not fully reflect clinical tumor physiology. Because cells grown in a 3-D configuration more closely resemble an in vivo environment, a model was developed and characterized for the growth of SK-Hep human hepatoma cells in suspension as multicellular tumor spheroids (MTS). The measurement of nutrient uptake in such a system has never been established. MATERIALS AND METHODS: SK-Hep cultures were initiated as single cell suspensions and grown as MTS in siliconized spinner flasks. The transport of several individual amino acids (arginine, glutamate, leucine, alpha-(N-methylamino)isobutyric acid (MeAIB), and glutamine (GLN)) was measured in SK-Hep single cell suspensions and MTS (0. 50-0.60 mm diameter) by a radiotracer/rapid filtration technique, as was the regulation of glutamine uptake by phorbol esters. l-[(3)H]GLN uptake was also measured in larger spheroids (0.85-1.5 mm diameter). MTS cellularity was evaluated by histological examination, and single cell integrity after the transport assay was confirmed by scanning electron microscopy (SEM). RESULTS: SK-Hep MTS displayed gradients of cellular morphology and staining, with central necrosis visible at diameters >0.8 mm. Single cell suspensions endured the rapid filtration technique based on functional Na(+)-dependent uptake rates and SEM analysis. Of all amino acids tested, only GLN transport rates were visibly affected by growth format. In small MTS, Na(+)-dependent GLN uptake was diminished by 40%, but was 40-53% higher in MTS >1 mm displaying central necrosis, when compared to single cell suspensions. Likewise, slight parallel changes in glutamine transporter ATB(0) mRNA levels were observed in Northern blot analysis. Finally, phorbol ester-dependent GLN transport down-regulation (by 40-50%), previously established in SK-Hep monolayers, remained operative in all cell formats tested. CONCLUSIONS: The data suggest that the tumor microenvironment differentially impacts the uptake of specific nutrients despite the conservation of key regulatory pathways. This MTS technique may prove useful for further studies on the role of nutrient transport in nascent tumor growth.

Amino Acid Transport System ASC↗

PubMed2000

DNA microarray detection of metabolic responses to protein overproduction in Escherichia coli.

It has been commonly observed that gratuitous overexpression of proteins in Escherichia coli causes growth retardation. However, the molecular events involved in the metabolic response to the over-expression of proteins are still unclear. Here we used DNA microarray technology to characterize the changes in transcriptional patterns of selected host genes during protein overexpression. A nontoxic, soluble protein, LuxA (coded by luxA), which is the alpha-subunit of the luciferase heterodimer, was overexpressed for this purpose. A total of 132 E. coli genes, including those in the central metabolism, key biosynthetic pathways, and selected regulatory functions, were used as probes for detecting the level of mRNA transcripts in E. coli strains JM109, MC4100, and VJS676A during protein overexpression. Upon induction, these strains shared several common responses, such as the upregulation of glk and the heat shock genes as well as the downregulation of fba, ppc, atpA, and gnd. In addition, the biosynthesis genes glnA, glyA, and leuA were downregulated in all three strains. Media-dependent responses were also observed in our study. For example, many respiratory genes that were upregulated in defined media showed an opposite effect in complex media under protein-overproducing conditions. These results demonstrate that gratuitous overexpression of proteins triggers a complex global response that involves several metabolic and regulatory systems. Explanations based on either existing knowledge of global regulations such as the heat shock response and the stringent response or stoichiometric analysis without regulatory considerations cannot account for the response induced by protein overexpression.

Bacterial Proteins↗