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PubMed1987

Glutathione depletion suppresses conjugation of benzo[a]pyrene metabolites and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites with glutathione but does not affect their binding to DNA in C3H/10T1/2 mouse fibroblasts.

The study was aimed at determining the role of glutathione (GSH) conjugation in the binding of reactive benzo[a]pyrene (BaP) species to DNA of C3H/10T1/2 cells. In order to suppress GSH conjugation cells were depleted of GSH by treatment with buthionine sulfoximine for 18 h and 1-chloro-2,4-dinitrobenzene for 1 h prior to incubation with radiolabelled substrates. Under these conditions GSH levels decreased to less than 1% of the control value. C3H/10T1/2 cells produced GSH conjugates with 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE) comprising 6% of the total metabolites formed from BaP or (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-diol). In GSH-depleted cells formation of GSH conjugates with metabolic products of BaP or BaP-7,8-diol was suppressed to 1% of total metabolites during an 8-h incubation period. Metabolic activation of BaP and BaP-7,8-diol by C3H/10T1/2 cells resulted in the formation of DNA adducts which largely consisted of BaPDE:deoxyguanosine. Depletion of GSH altered neither the degree of DNA binding nor the pattern of DNA adducts to any significant extent. When C3H/10T1/2 cells were co-incubated with microsomes from liver of 3-methylcholanthrene-treated rats for 1 h in order to activate BaP or BaP-7,8-diol extracellularly, the same pattern of GSH conjugates and DNA adducts was generated as by intracellular metabolism of the polycyclic hydrocarbons. No GSH conjugates were detected following co-incubation of microsomes with GSH-depleted C3H/10T1/2 cells. The formation of DNA adducts again remained unaffected by the suppression of conjugation. C3H/10T1/2 cells are apparently capable of conjugating BaPDE with GSH but are not capable of trapping by GSH conjugation those BaPDE moieties which bind to DNA. The results are compatible with the notion that BaPDE is partially contained in a cellular compartment--presumably the lipid environment of membranes--where it is inaccessible to GSH transferases of C3H/10T1/2 cells.

Animals↗

PubMed1980

The site of cartilage matrix degradation.

1. A 'cavity' organ culture method was developed, which allowed the insertion within bovine nasal cartilage of 10 microliter of 35S-labelled proteoglycan aggregate. 2. Stimulation of the tissue with retinol or catabolin led to degradation of both the cartilage matrix and the proteoglycan within the cavity. 3. Proteoglycan aggregate trapped in polyacrylamide beads was not broken down in the cavities of living cartilage, except under autolytic conditions. 4. It is suggested that degradation of proteoglycan aggregate by chondrocytes occurs in a pericellular region.

Acrylamides↗

PubMed1998

Lysosomal routing of Fc gamma RI from early endosomes requires recruitment of tyrosine kinases.

The high-affinity receptor for immunoglobulin G (Fc gamma RI) plays a central role in the clearance of immune complexes by mediating their internalization and delivery to lysosomes. In monocytic U937 cells, receptor internalization is independent of tyrosine kinase activity. However, the tyrosine kinase inhibitor, genistein, prevents further progress of the receptor to lysosomes and traps it in a sub-plasma membrane early endosome. Similarly, Fc gamma RI expressed in COS cells is able to internalize immune complexes but is unable to translocate to lysosomes. This suggests that Fc gamma RI, whose cytoplasmic tail is devoid of known signalling motifs, must recruit tyrosine kinases via its gamma-chain to achieve lysosomal delivery. We show that a chimera of the extracellular domain of Fc gamma RI and the cytoplasmic tail of the gamma-chain is both internalized and efficiently trafficked to lysosomes. Our study suggests that a key function of the gamma-chain is recruitment of tyrosine kinases to initiate the intracellular signalling pathways required to target Fc gamma RI following immune complex aggregation to lysosomes and not to initiate endocytosis per se.

Antigen-Antibody Complex↗

PubMed2004

Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approach.

In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo.

Amino Acid Sequence↗

PubMed1995

In vitro refolding of cyclomaltodextrin glucanotransferase from cytoplasmic inclusion bodies formed upon expression in Escherichia coli.

The recovery of active cyclomaltodextrin glucanotransferase (CGT) from inclusion bodies formed upon expression of a signal sequence deletion mutant (cgt delta ss) gene in Escherichia coli was studied. Under the conditions examined the in vitro renaturation yield of active enzyme was up to 81% of the maximum activity from urea solubilized and up to 3.7% from guanidinium hydrochloride (GdmCl) solubilized inclusion bodies. Refolding from GdmCl solutions resulted in the reaggregation of delta ss CGT. Although native (folded) CGT showed enzymatic activity at urea concentrations as high as 10 M, the inclusion bodies could be solubilized with 3 M urea solution, but the preparation had no enzymatic activity prior to reduction of the denaturant concentration. This suggests that the delta ssCGT inclusion bodies are composed of molecules that are trapped in an inactive state. The individual molecules may have extensive secondary structure, since inclusion bodies solubilized with 4.5 M urea gave maximum yield of activity in the renaturation step. With higher urea concentration the yield decreased. Thus, a "mild" solubilization technique seems to increase the yield of active delta ssCGT from the inclusion bodies. The stability of the refolded delta ssCGT was identical to that of extracellularly produced wild type CGT, whereas a disulfide bond mutant, Ser-70-CGT, showed reduced stability under identical conditions. This suggests that the single disulfide bond in delta ssCGT is formed during refolding and contributes to the stability of the molecule. A fusion of maltose binding protein to the NH2-terminus of delta ssCGT did not significantly affect the in vitro folding of the delta ssCGT portion when tested by a denaturation-renaturation experiment.

ATP-Binding Cassette Transporters↗

PubMed2002

Regulative function of telomerase and extracellular regulated protein kinases to leukemic cell apoptosis.

In order to investigate the regulative function of telomerase and phosphorylated (activated) extracellular regulated protein kinase (ERK) 1 and 2 in the leukemic cell lines HL-60 and K562 proliferation inhibition and apoptosis, three chemotherapeutic drugs Harringtonine (HRT), Vincristine (VCR) and Etoposide (Vp16) were selected as inducers. The proliferation inhibition rate was detected by MTT method, the cell cycle and cell apoptosis was analyzed by flow cytometry and the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis method. The phosphorylated ERK1/2 protein expression was detected by western blot method. The results showed that HRT, VCR and Vp16 could inhibit cell proliferation, induce apoptosis, inhibit telomerase activity and down-regulate the protein expression of phosphorylated ERK. It was suggested that ERK signal transduction pathway was involved in the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treated by HRT, VCR and Vp16.

Antineoplastic Agents↗

PubMed2004

Separation of individual neurons using dielectrophoretic alternative current fields.

Experimental investigations into the dynamics of neuronal networks are a fundamental step towards understanding how the nervous system works. Memory formation and development are associated with changes in the electrical activity of the neurons. To understand the changes in the electrical activity, it is essential to conduct in vitro studies on individual neurons. Hence, there is an enormous need to develop novel ways for isolating and localizing individual neurons. To this end, we designed and fabricated a 4x4 multiple microelectrode array system to spatially arrange neurons by generating dielectrophoretic traps using gradient alternating current (AC) fields. We characterized the electric field distribution inside our test platform by using three-dimensional finite element modeling (FEM) and estimated the location of neurons over the electrode array. As the first stage in forming a neuronal network, dielectrophoretic AC fields were employed to separate the neurons from the glial cells and to position individual neurons over single electrodes. The extracellular electrical activity from a single neuron was recorded. The frequency spectrum of the electrical activity was generated using fast Fourier transformation analysis (FFT) to determine the characteristic burst rates of individual neurons.

Action Potentials↗

PubMed2006

Tamoxifen protect against hydroxyl radical generation induced by phenelzine in rat striatum.

The present study was examined whether tamoxifen, a synthetic nonsteroidal antiestrogen, could suppress antidepressant drug phenelzine can increase an active dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+))-induced hydroxyl radical (OH) generation in the extracellular fluid of rat striatum, using in vivo microdialysis system. Rats were anesthetized, and sodium salicylate (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Infusion of phenelzine (100 microM or 0.1 nmol/microl/min) into the striatum drastically increased dopamine (DA) efflux and the OH formation, trapped as 2,3-DHBA by the possible increased production of MPP(+). However, tamoxifen (100 microM) significantly suppressed phenelzine enhanced DA efflux and OH formation by MPP(+). These results in the present study is the first demonstration showing the protective effect of tamoxifen on OH generation induced by phenelzine enhanced MPP(+) by suppressing DA efflux.

1-Methyl-4-phenylpyridinium↗

PubMed2001

ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide.

We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O2*- generation during phagocytosis in human polymorphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zymosan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of Cu,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-adducts were present, cells after stimulation were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), the DEPMPO-OOH was detected in extracellular fluid but not in the cell fraction. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O2*- of extracellular space including the intraphagosome but not that of intracellular space in sOZ-stimulated phagocytes.

Cyclic N-Oxides↗

PubMed1962

The active transport of sodium by ghosts of human red blood cells.

The outflux of Na(24) from prelabeled ghosts was measured under various conditions. Prelabeling was accomplished by hypotonic hemolysis of intact human cells in the presence of tracer Na(24). The resultant ghosts when subsequently washed were found to retain 10 to 20 per cent of the initial Na(24). Separate experiments indicated that this trapped amount resides in only a portion of ghosts comprising the total population. The characteristics of the outflux of this residual Na(24) indicated that the ghost system closely resembles intact red cells. The outflux of Na from ghosts could be divided into three components: active and passive transport and exchange diffusion. The active transport system, necessarily driven by metabolism, required the presence of K in the extracellular phase and was blocked by strophanthidin. The concentration dependence of the Na pump flux on the external K and internal Na appeared the same in ghosts as in intact cells. Certain other features of this ghost system are also discussed.

Biological Transport↗

PubMed2002

Disruption of the gene encoding the latent transforming growth factor-beta binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer.

Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Spin trapping study in the lungs and liver of F344 rats after exposure to ozone.

Fischer 344 rats were injected with the spin traps C-phenyl N-tert-butyl nitrone (PBN, 150 mg/kg bw, ip) or 4-pyridine-N-oxide N-tert-butyl nitrone (POBN, 775 mg/kg bw, ip), and exposed to clean air or 2 ppm ozone for two hours. The presence of spin adducts was determined by electron paramagnetic resonance (EPR) spectroscopy of chloroform extracts of lung and liver homogenates. No significant levels of adducts were detected in the lungs of air control animals. Benzoyl N-tert-butyl aminoxyl, attributed to direct reaction of ozone with PBN, and tert-butyl hydroaminoxyl, the scission product of the hydroxyl adduct of PBN, were detected in the lungs of ozone exposed rats. EPR signals for carbon-centred alkoxyl and alkyl adducts were also detected with PBN in the lungs and liver of animals exposed to ozone. With POBN, only carbon-centred alkyl radicals were detected. Senescent, 24 months old rats were found to retain about twice more 14C-PBN in blood, heart and lungs by comparison to juvenile, 2 months old animals. Accordingly, the EPR signals were generally stronger in the lungs of the senescent rats by comparison to juvenile rats. Together, the observations were consistent with the previously proposed notion that a significant flux of hydrogen peroxide produced from the reaction of ozone with lipids of the extracellular lining, or from activated macrophages in the lungs could be a source of biologically relevant amounts of hydroxyl radical.

Animals↗

PubMed2004

Effects of the phytoestrogen coumestrol on RANK-ligand-induced differentiation of osteoclasts.

Phytoestrogens, which have structural similarity to 17beta-estradiol, have been reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to have an important role in maintaining bone mass, because the concentration of serum estrogen decreases after menopause and the estrogen deficiency causes bone loss. In this study, we investigated the effects of coumestrol and other phytoestrogens on osteoclast differentiation using estrogen receptor alpha-transfected RAW264.7 (RAW264.7-ERalpha) cells. When the cells were cultured with the receptor activator of nuclear factor kappa B-ligand (RANKL), both formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and TRAP activity were increased compared with control cells that were cultured in the absence of RANKL. Coumestrol decreased RANKL-induced formation of TRAP-positive multinucleated cells and TRAP activity dose-dependently. RANKL-stimulated RAW264.7-ERalpha cells formed resorption pits on calcium phosphate films and the pit formation was inhibited by coumestrol in a dose-dependent manner. RT-PCR analyses revealed that coumestrol (10 microM) decreased mRNA levels of calcitonin receptor (CTR) and matrix metalloproteinase-9 (MMP9) in RANKL-treated cells. In addition, pretreatment of coumestrol decreased RANKL-induced phosphorylation of extracellular signal-regulated kinases/p44/42 (ERK1/2). These results suggest that coumestrol has an inhibitory effect on the differentiation of osteoclasts, at least partially via ERK1/2 pathway.

Acid Phosphatase↗

PubMedDate not supplied

X-ray diffraction of bacteriorhodopsin photocycle intermediates.

Recent advances in the crystallography of bacteriorhodopsin, the light-driven proton pump, have yielded structural models for all intermediates of the photochemical cycle. For seven of the species, X-ray diffraction data were collected from trapped photostationary states in crystals, and for the two remaining ones the structures of selected mutants are available. The changes of the retinal chromophore, protein and bound water describe, at an atomic level, how accommodation of the twisted photoisomerized retinal to its binding site causes de-protonation of the retinal Schiff base and initiates cascades of gradual conformational rearrangements of the protein. One cascade propagates in the extracellular direction and results in proton release, and the other in the cytoplasmic direction and results in side-chain and main-chain rearrangements, formation of a chain of hydrogen-bonded water, and proton uptake from the bulk. Such local-global conformational coupling, with gradual spreading of a local perturbation over the rest of the protein, might be the uniting principle of transporters and receptors.

Bacteriorhodopsins↗

PubMed1988

Uptake of erythromycin by McCoy and HEp2 cells: its dependence on cellular pH gradients.

The accumulation of erythromycin, a weak lipophilic base, was studied in two mammalian cell lines. Uptake was similar in both cell types, being highly pH dependent and not saturated even at external concentrations of 1000 mg/l. At pH 6.7 the ratio of cellular:extracellular erythromycin concentrations (C/E) was only 2.0, whereas at pH 7.5 it was as high as 7.0. Carbonylcyanide-m-chlorophenyl hydrazone, an uncoupler of oxidative phosphorylation which was used to collapse cellular pH gradients, caused a 50% reduction in C/E ratio within five minutes when the external pH was greater than 6.9 but had no effect at or below pH 6.7. These results suggest that ion-trapping plays a major role in the accumulation of erythromycin by these cells.

Cell Line↗

PubMed1991

Regulation of cytosolic pH of cultured mesangial cells by prostaglandin F2 alpha and thromboxane A2.

Prostaglandins (PG) and thromboxane A2 (TxA2) have marked vasoactive effects on the renal glomerular microcirculation. Exposure of cultured mesangial cells to PGF2 alpha and TxA2 mimetics results in a rapid elevation of free cytosolic Ca2+ ([Ca2+]i) followed by contraction and cell proliferation. We studied whether other ionic changes mediate these effects of eicosanoids on cells of rat and human origin. Cytoplasmic pH (pHi) was monitored in cells loaded with the fluorescent, intracellularly trapped pH-sensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. PGF2 alpha in rat cells and the TxA2 mimetic U-46619 in human cells induced rapid, dose-dependent cytosolic acidification followed by recovery and net alkalinization mediated by enhanced Na(+)-H+ exchange. The early acidification was also stimulated by ionomycin and Ca2(+)-mobilizing peptides, implicating a Ca2(+)-dependent mechanism. Alkalinization was abolished by removal of extracellular Na+ and by amiloride. Both components of the responses were inhibited by phorbol myristate acetate, which could mimic alkalinization, suggesting a regulatory role of protein kinase C in activation of the Na(+)-H+ exchanger by eicosanoids. Vasoconstrictor arachidonate metabolites may control glomerular cell function by a signaling mechanism centered on concurrent changes of pHi and [Ca2+]i.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1977

Transport and metabolism of vitamin B6 in lactic acid bacteria.

Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.

Biological Transport, Active↗

PubMed2006

Identification of novel cell-adhesion molecules in peripheral nerves using a signal-sequence trap.

The development and maintenance of myelinated nerves in the PNS requires constant and reciprocal communication between Schwann cells and their associated axons. However, little is known about the nature of the cell-surface molecules that mediate axon-glial interactions at the onset of myelination and during maintenance of the myelin sheath in the adult. Based on the rationale that such molecules contain a signal sequence in order to be presented on the cell surface, we have employed a eukaryotic-based, signal-sequence-trap approach to identify novel secreted and membrane-bound molecules that are expressed in myelinating and non-myelinating Schwann cells. Using cDNA libraries derived from dbcAMP-stimulated primary Schwann cells and 3-day-old rat sciatic nerve mRNAs, we generated an extensive list of novel molecules expressed in myelinating nerves in the PNS. Many of the identified proteins are cell-adhesion molecules (CAMs) and extracellular matrix (ECM) components, most of which have not been described previously in Schwann cells. In addition, we have identified several signaling receptors, growth and differentiation factors, ecto-enzymes and proteins that are associated with the endoplasmic reticulum and the Golgi network. We further examined the expression of several of the novel molecules in Schwann cells in culture and in rat sciatic nerve by primer-specific, real-time PCR and in situ hybridization. Our results indicate that myelinating Schwann cells express a battery of novel CAMs that might mediate their interactions with the underlying axons.

Journal Article↗