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PubMed2002

Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17beta-estradiol.

Little is known about the regulation of cytosolic calcium Ca(2+) levels ([Ca(2+)](i)) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF-7 and its responsiveness to ATP. MCF-7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca(2+), resulted in a rapid and transient elevation in [Ca(2+)](i). After recovery of basal levels, Ca(2+) readmission (1.5 mM) to the medium increased Ca(2+) influx (twofold over basal), reflecting pre-activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca(2+) store is involved in their response. Moreover, IP(3)-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, an inhibitor of phospholipase C. Compound 2-APB (75 microM) and Gd(3+) (10 microM), antagonists of the CCE pathway, completely prevented ATP-stimulated capacitative Ca(2+) entry. CCE in MCF-7 cells was highly permeable to Mn(2+) and to the Ca(2+) surrogate Sr(2+). Mn(2+) entry sensitivity to Gd(3+) matched that of the Ca(2+) entry pathway. 17Beta-estradiol blocked ATP-induced CCE, but was without effect on TG-induced CCE. Besides, the estrogen blockade of the ATP-induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER alpha immunoreactivity could also be detected in a purified plasma membrane fraction of MCF-7 cells. These results represent the first evidence on the operation of a ATP-responsive CCE pathway in MCF-7 cells and also indicate that 17beta-estradiol interferes with this mechanism by acting at the cell surface level.

Adenosine Triphosphate↗

PubMed2003

2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels.

The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca(2+) signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOC). In addition to abrogating hormone-evoked Ca(2+) responses, 2-APB could antagonise Ca(2+) signals evoked by a membrane permeant InsP(3) ester. 2-APB also slowed the recovery of intracellular Ca(2+) signals consistent with an effect on Ca(2+) ATPases. The inhibitory action of 2-APB on InsP(3) receptors (InsP(3)Rs), SOC channels and Ca(2+) pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca(2+) responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca(2+) channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca(2+) signalling in electrically-stimulated cardiac myocytes, where InsP(3)Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca(2+) signalling in cell types where InsP(3) and SOC are not active.

Boron Compounds↗

PubMed2006

Electrochemical degradation and toxicity reduction of C.I. Basic Red 29 solution and textile wastewater by using diamond anode.

Electrochemical oxidation of Basic Red 29 (BR29) was studied in a bipolar trickle tower (BTT) reactor by using Raschig ring shaped boron-doped diamond (BDD) electrodes, which were originally employed by the present researchers, in a recirculated batch mode. The model solution was prepared with BR29 using distilled water. The effects of initial dye concentration, Na(2)SO(4) concentration as supporting electrolyte, current density, flow rate and initial pH on the removal efficiency were investigated, and practically, complete BR29 removal (over 99%) was obtained in all the studies. After optimum experimental conditions were determined, textile wastewater has also studied by monitoring the destruction of color and COD. With the textile wastewater, 97.2% of color and 91% of COD removal were, respectively, achieved at the current density of 1mA/cm(2). Microtox toxicity tests were performed in both BR29 solution and textile wastewater under optimum experimental conditions, and relatively good toxicity reductions were obtained with respect to the initial values. According to the results, BDD anode was seen to be a unique material for the degradation of BR29 and COD and also the reduction of toxicity simultaneously.

Aliivibrio fischeri↗

PubMed1989

envM genes of Salmonella typhimurium and Escherichia coli.

Conjugation and bacteriophage P1 transduction experiments in Escherichia coli showed that resistance to the antibacterial compound diazaborine is caused by an allelic form of the envM gene. The envM gene from Salmonella typhimurium was cloned and sequenced. It codes for a 27,765-dalton protein. The plasmids carrying this DNA complemented a conditionally lethal envM mutant of E. coli. Recombinant plasmids containing gene envM from a diazaborine-resistant S. typhimurium strain conferred the drug resistance phenotype to susceptible E. coli cells. A guanine-to-adenine exchange in the envM gene changing a Gly codon to a Ser codon was shown to be responsible for the resistance character. Upstream of envM a small gene coding for a 10,445-dalton protein was identified. Incubating a temperature-sensitive E. coli envM mutant at the nonpermissive temperature caused effects on the cells similar to those caused by treatment with diazaborine, i.e., inhibition of fatty acid, phospholipid, and lipopolysaccharide biosynthesis, induction of a 28,000-dalton inner membrane protein, and change in the ratio of the porins OmpC and OmpF.

Alleles↗

PubMed2002

Role of RyRs and IP3 receptors after traumatic injury to spinal cord white matter.

Calcium influx and elevation of intracellular free calcium (Ca2+i), with subsequent activation of degenerative enzymes is hypothesized to cause cell injury and death after trauma. We examined the effects of traumatic compressive injury on (Ca2+)i dynamics in spinal cord white matter. We conducted electrophysiological studies with ryanodine and inositol (1,4,5)-triphosphate (IP3) receptor agonists and antagonists in an in vitro model of spinal cord injury (SCI). A 25-30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber (37 degrees C) and injured with a modified clip (2-g closing force) for 15 sec. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential (CAP) with glass microelectrodes. The CAP decreased to 55.2+/-6.8% of control (p < 0.05) after spinal cord injury (SCI). Chelation of Ca2+i with BAPTA-AM (a high-affinity calcium chelator) promoted significantly greater recovery of CAP amplitude (83.2+/-4.2% of control; p < 0.05) after injury. Infusion of caffeine (1 and 10 mM) exacerbated CAP amplitude decline (45.1+/-5.9% of control; p < 0.05; 44.6+/-3.1% of control; p < 0.05) postinjury. Blockade of Ca2+i release through ryanodine-sensitive receptors (RyRs) with dantrolene (10 microM) and ryanodine (50 microM), conferred significant (p < 0.05) improvement in CAP amplitude after injury. On the other hand, blockade of Ca2+i with inositol (1,4,5)-triphosphate receptor (IP3Rs) blocker 2APB (10 microM) also conferred significant improvement in CAP amplitude after injury (82.9+/-7.9%; p < 0.05). In conclusion, the injurious effects of Ca2+i in traumatic central nervous system (CNS) white matter injury appear to be mediated both by RyRs and through IP3Rs calcium-induced calcium release receptors (CICRs).

Action Potentials↗

PubMed1995

Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation.

In situ presence of numerous DNA strand breaks is a typical feature of apoptotic cells. Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated DNA precursors has recently been proposed as a method of analysing DNA replication. Detection of DNA strand breaks, thus, enables one to identify apoptotic and/or DNA replicating cells. The current methods for DNA strand break labelling rely on the use of exogenous terminal deoxynucleotidyl transferase which either directly attaches the fluorochrome conjugated triphosphodeoxynucleotides to 3'OH ends in the breaks, or indirectly labels 3'OH ends with digoxygenin or biotin conjugated triphosphodeoxynucleotides. A limitation of these methodologies, especially restricting their routine application in the clinic, is high cost of reagents. In the present study we have tested whether relatively simple compound BrdUTP, which is approximately three orders of magnitude less expensive than dUTP conjugated to digoxygenin, can be used as marker of DNA strand breaks. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin. The incorporated BrdUTP was detected by fluoresceinated anti-BrdUrd MoAb. Cellular fluorescence was measured by flow cytometry as well as by Laser Scanning Cytometer (LSC). The data show that intensity of DNA strand break labelling with BrdUTP was nearly four- and two-fold higher than that obtained with the indirect labelling using biotin- or digoxygenin-conjugated dUTP, respectively, and over eight-fold higher than in the case of direct labelling with the fluorochrome (fluorescein or BODIPY)-conjugated deoxynucleotides. The increased labelling of DNA strand breaks with BrdUTP may reflect more efficient incorporation of this precursor by terminal transferase, compared to the nucleotides with bulky fluorochrome conjugates. DNA strand break labelling with BrdUTP, thus, offers a possibility of more sensitive (and at lower cost) detection of apoptotic or DNA replicating cells, compared to the alternative methods of DNA strand break labelling.

Apoptosis↗

PubMed1988

The influence of tetraphenylborates (hydrophobic anions) on yeast cell electro-rotation.

The action of a series of tetraphenylborate ion (TPB) derivatives on yeast cells was studied by electro-rotation of the pre-treated cells. TPB derivatives in which all four phenyl groups were substituted with fluorine, chlorine or trifluoromethyl were much more toxic than the unsubstituted compound, the effect increasing dramatically with increasing size of substituents. These observations suggest that the toxicity of these hydrophobic ions is determined mainly by their size and possibly also by the chemical inductivity of their substituent groups. The order of the toxicities of these ions was in fair agreement with literature values for their translocation rates across artificial bilayers. Incubation times of 3 h were used as standard, longer incubations (up to 48 h) showed that the number of cells affected by low doses of TPB increased with the logarithm of time after the first hour of incubation. Although measurements of the percentage of cells showing co-field rotation showed that controls were not adversely affected by incubations as long as 9 h, rotation spectra showed that some cells suffer loss of internal conductivity during extended incubations. Decrease of the pH of the incubation medium, or inclusion of high concentrations of NaCl or KCl, potentiated the effects of these hydrophobic ions. The toxicity developed slowly, and the sensitivity of the assay was only very weakly dependent on the cell suspension density.

Boron Compounds↗

PubMed1997

Synthesis and characterization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled fluorescent ligands for the mu opioid receptor.

A series of opioid ligands utilizing the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid or 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid were synthesized and characterized for their ability to act as a suitable fluorescent label for the mu opioid receptor. All compounds displaced the mu opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with high affinity. The binding of fluorescent ligands to delta and kappa receptors was highly variable. 5,7-Dimethyl-BODIPY naltrexamine, "6-BNX," displayed subnanomolar affinities for the mu and kappa opioid receptors (Ki 0.07 and 0.43 nM, respectively) and nanomolar affinity at the delta (Ki 1.4 nM) receptor. Using fluorescence spectroscopy, the binding of 6-BNX in membranes from C6 glioma cells transfected with the cloned mu opioid receptor was investigated. In these membranes containing a high receptor density (10-80 pmol/mg protein), 6-BNX labeling was saturable, mu opioid specific, stereoselective (as determined with the isomers dextrorphan and levorphanol), and more than 90% specific. The results describe a series of newly developed fluorescent ligands for the mu opioid receptor and the use of one of these ligands as a label for the cloned mu receptor. These ligands provide a new approach for studying the structural and biophysical nature of opioid receptors.

Animals↗

PubMed2003

Localised calcium release events in cells from the muscle of guinea-pig gastric fundus.

After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.

Animals↗

PubMed1999

Synthesis of 5-(carboranylalkylmercapto)-2'-deoxyuridines and 3-(carboranylalkyl)thymidines and their evaluation as substrates for human thymidine kinases 1 and 2.

Derivatives of thymidine containing o-carboranylalkyl groups at the N-3 position and derivatives of 2'-deoxyuridine containing o-carboranylalkylmercapto groups at the C-5 position were synthesized. The alkyl spacers consist of 4-8 methylene units. The synthesis of the former compounds required 3-4 reaction steps in up to 75% overall yield and that of the latter 9-10 reaction steps with significantly lower overall yield. Derivatives of thymidine substituted with carboranylalkyl substituents at the N-3 position and short spacers were phosphorylated by both recombinant and purified cytosolic thymidine kinase (TK1) to a relatively high degree. None of the tested 2'-deoxyuridine derivatives possessing carboranyl substituents at the C-5 position were phosphorylated by either recombinant or purified TK1. The amounts of phosphorylation product detected for some of the C-5-substituted nucleosides with recombinant mitochondrial thymidine kinase (TK2) were low but significant and decreased with increasing lengths of the alkyl spacer. The data obtained in this study do not seem to support the tether concept applied in the synthesis of the new C-5- and N-3-substituted carboranyl nucleosides intended to reduce possible steric interference in the binding of carboranyl nucleosides with deoxynucleoside kinases. Instead, it appeared that a closer proximity of the bulky carborane moiety to the nucleoside scaffold resulted in better substrate characteristics.

Boron Compounds↗

PubMed1996

The enoyl-[acyl-carrier-protein] reductase (FabI) of Escherichia coli, which catalyzes a key regulatory step in fatty acid biosynthesis, accepts NADH and NADPH as cofactors and is inhibited by palmitoyl-CoA.

Reduction of enoyl-acyl-carrier-protein (ACP) substrates by enoyl-ACP reductase is a key regulatory step in fatty acid elongation of Escherichia coli. Two enoyl-ACP reductase activities have been described in E. coli, one specific for NADH, the other for NADPH as cofactor. Because of their distinct enzymatic properties, these activities were ascribed to two different proteins. The NADH-dependent enoyl-ACP reductase of E. coli has previously been identified as the FabI protein, which is the target of a group of antibacterial compounds, the diazaborines. We now demonstrate that both enoyl-ACP reductase activities reside in FabI. In crude cell extracts of FabI-overproducing strains, both NADH-dependent and NADPH-dependent enoyl-ACP reductase activities are increased. Mutations in the fabI gene that lead either to temperature-sensitive growth or diazaborine resistance result in the reduction of both activities. When FabI is purified in pH 6.5 buffers, the protein exhibits NADH-dependent and NADPH-dependent reductase activities. Both enzymatic activities are inhibited by diazaborine. The NADPH-dependent enoyl-ACP reductase activity, however, turned out to be approximately eight times more resistant to diazaborine. The difference in sensitivity indicates that binding of either NADPH or NADH to FabI results in distinct changes in the configuration of the protein or, alternatively, it is different due to the different charge of the cofactors. These effects might be responsible for the differences in the enzymatic properties. Both reductase activities of the FabI protein are inhibited by physiologically relevant concentrations of palmitoyl-CoA, which might be important in regulating endogenous fatty acid biosynthesis in E. coli in the presence of exogenous fatty acids.

Anti-Bacterial Agents↗

PubMed2005

Biphasic currents evoked by chemical or thermal activation of the heat-gated ion channel, TRPV3.

2-Aminoethyl diphenylborinate was recently identified as a chemical activator of TRPV1, TRPV2, and TRPV3, three heat-gated members of the transient receptor potential vanilloid (TRPV) ion channel subfamily. Here we demonstrated that two structurally related compounds, diphenylboronic anhydride (DPBA) and diphenyltetrahydrofuran (DPTHF), can also modulate the activity of these channels. DPBA acted as a TRPV3 agonist, whereas DPTHF exhibited prominent antagonistic activity. However, all three diphenyl-containing compounds promoted some degree of channel activation or potentiation, followed by channel block. Strong TRPV3 activation by DPBA often leads to the appearance of a secondary, enhanced, current phase. A similar biphasic response was observed during TRPV3 heat stimulation; an initial, gradually sensitizing phase (I(1)) was followed by an abrupt transition to a secondary phase (I(2)). I(2) was characterized by larger current amplitude, loss of outward rectification, and alterations in the following properties: permeability among cations; ruthenium red and DPTHF sensitivity; temperature dependence; and voltage-dependent gating. The I(1) to I(2) transition depended strongly on TRPV3 current density. Removal of extracellular divalent cations resulted in heat-evoked currents resembling I(2), whereas mutation of a putative Ca(2+)-binding residue in the pore loop domain, aspartate 641, facilitated detection of the I(1) to I(2) transition, suggesting that the conversion to I(2) resulted from the agonist- and time-dependent loss of divalent cationic inhibition. Primary keratinocytes overexpressing exogenous TRPV3 also exhibited biphasic agonist-evoked currents. Thus, strong activation by either chemical or thermal stimuli led to biphasic TRPV3 signaling behavior that may be associated with changes in the channel pore.

Animals↗

PubMed1989

Treatment of malignant melanoma by selective thermal neutron capture therapy using melanoma-seeking compound.

As pigment cells undergo melanoma genesis, accentuated melanogenesis concurrently occurs in principle. Subsequent to the understanding of intrinsic factors controlling both processes, we found our selective melanoma neutron capture therapy (NCT) using 10B-dopa (melanin substrate) analogue, 10B1-p-boronophenylalanine (10B1-BPA), followed by 10B(n, alpha)7Li reaction, induced by essentially harmless thermal neutrons, which releases energy of 2.33 MeV to 14 mu, the diameter of melanoma cells. In vitro/in vivo radiobiological analysis revealed the highly enhanced melanoma killing effect of 10B1-BPA. Chemical and prompt gamma ray spectrometry assays of 10B accumulated within melanoma cells after 10B1-BPA administration in vitro and in vivo show high affinity, e.g., 10B melanoma/blood ratio of 11.5. After successfully eradicating melanoma transplanted into hamsters with NCT, we advanced to preclinical studies using spontaneously occurring melanoma in Duroc pig skin. We cured three melanoma cases, 4.6 to 12 cm in diameter, by single neutron capture treatment. Complete disappearance of melanoma was obtained without substantial side effects. Acute and subacute toxicity as well as pharmacodynamics of 10B1-BPA have been studied in relation to therapeutic dosage requirements. Clinical radiation dosimetry using human phantom has been carried out. Further preclinical studies using human melanoma transplanted into nude mouse have been a useful model for obtaining optimal results for each melanoma type. We recently treated the first human melanoma patient with our NCT, using essentially the method for Duroc pig melanoma, and obtained similar regression time course leading to cure.

Animals↗

PubMed2006

Regulation of CRAC channel activity by recruitment of silent channels to a high open-probability gating mode.

CRAC (calcium release-activated Ca(2+)) channels attain an extremely high selectivity for Ca(2+) from the blockade of monovalent cation permeation by Ca(2+) within the pore. In this study we have exploited the blockade by Ca(2+) to examine the size of the CRAC channel pore, its unitary conductance for monovalent cations, and channel gating properties. The permeation of a series of methylammonium compounds under divalent cation-free conditions indicates a minimum pore diameter of 3.9 A. Extracellular Ca(2+) blocks monovalent flux in a manner consistent with a single intrapore site having an effective K(i) of 20 microM at -110 mV. Block increases with hyperpolarization, but declines below -100 mV, most likely due to permeation of Ca(2+). Analysis of monovalent current noise induced by increasing levels of block by extracellular Ca(2+) indicates an open probability (P(o)) of approximately 0.8. By extrapolating the variance/mean current ratio to the condition of full blockade (P(o) = 0), we estimate a unitary conductance of approximately 0.7 pS for Na(+), or three to fourfold higher than previous estimates. Removal of extracellular Ca(2+) causes the monovalent current to decline over tens of seconds, a process termed depotentiation. The declining current appears to result from a reduction in the number of active channels without a change in their high open probability. Similarly, low concentrations of 2-APB that enhance I(CRAC) increase the number of active channels while open probability remains constant. We conclude that the slow regulation of whole-cell CRAC current by store depletion, extracellular Ca(2+), and 2-APB involves the stepwise recruitment of silent channels to a high open-probability gating mode.

Boron Compounds↗

PubMed2005

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors.

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.

Biological Transport↗

PubMed2000

Nonpeptide factor Xa inhibitors II. Antithrombotic evaluation in a rabbit model of electrically induced carotid artery thrombosis.

SK549 (mol. wt. 546 Da) is a synthetic, selective inhibitor of human coagulation factor Xa (fXa) (K(i) = 0.52 nM). This study compared the antithrombotic effects of SK549 and a series of benzamidine isoxazoline fXa inhibitors with aspirin, DuP 714 (a direct thrombin inhibitor), recombinant tick anticoagulant peptide, or heparin in a rabbit model of electrically induced carotid arterial thrombosis. Compounds were infused i.v. continuously from 60 min before electrical stimulation to the end of the experiment. Values of ED(50) (dose that increases the carotid blood flow to 50% of the control) were 0.12 micromol/kg/h for SK549, 0.56 micromol/kg/h for aspirin, 0.14 micromol/kg/h for DuP 714, 0.06 micromol/kg/h for recombinant tick anticoagulant peptide, and >100 U/kg/h for heparin. The EC(50) (plasma concentration that increased blood flow to 50% of the control) for SK549 was 97 nM. Unlike aspirin and heparin, SK549 was efficacious and, at 1.5 micromol/kg/h i.v. (n = 9), maintained carotid blood flow at 87 +/- 6% of control level for greater than 90 min. Unlike heparin, SK549 inhibited ex vivo fXa activity but not ex vivo thrombin activity. There was a highly significant correlation between K(i) (fXa) and ED(50) of a series of fXa inhibitors (r = 0. 85, P <.001). Therefore, these results suggest that SK549 is a novel, potent, and effective antithrombotic agent in a rabbit model of arterial thrombosis. It is likely that SK549 exerts its antithrombotic effect through selective inhibition of fXa. Furthermore, SK549 may be clinically useful for the prevention of arterial thrombosis.

Animals↗

PubMed1996

Brefeldin A and mannose 6-phosphate regulation of acrosomic related vesicular trafficking.

Acrosomal biogenesis represents a unique system for the molecular analysis of the various processes involved in vesicular membrane transport. To assess the basic membrane trafficking mechanisms used in spermatids, we have used two fluorescent lipid compounds that label: a) the Golgi and Golgi-derived vesicles (C5-DMB-Cer), and b) endocytic vesicles (FM4-64). Incubation of mouse testicular germ cells at 33 degrees C for 1.5 h with C5-DMB-Cer revealed that C5-DMB-Cer labeling is localized in the Golgi and acrosome of early-maid round spermatid stages, with no labeling of the acrosome in late round spermatid stages. Culturing germ cells with FM4-64 for 1.5 h at 33 degrees C, showed that FM4-64 labeling in spermatids was localized in endocytic vesicles and Golgi of early-mid round spermatid stages, whereas in a small population of late round spermatid stages, FM4-64 was also localized in the apex region of the acrosome. Incubation with brefeldin A (BFA) (5 micrograms/ml) inhibited the distribution of C5-DMB-Cer and FM4-64 to the acrosome, however, it did not affect the localization of acrosin-an acrosome-specific protein-indicating that there was no apparent acrosome disassembly in the presence of BFA. Localization of FM4-64 in late round spermatids in the presence of 2.5 mM mannose 6-phosphate was found in endocytic vesicles and the Golgi, but not the acrosome. These results show that there are at least two sources of vesicular transport to the acrosome derived from the Golgi and plasma membrane.

Acrosin↗

PubMed2006

Application of lipid peroxidation and protein oxidation biomarkers for oxidative damage in mammalian cells. A comparison with two fluorescent probes.

We recently developed two biomarker sets for oxidative damage: one for determination of lipid peroxidation (LPO) degradation products; acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal, malondialdehyde and acetone, by a gas chromatography-electron capture detection method, and the other for protein oxidation products such as o,o'-dityrosine, by an isotope dilution high performance liquid chromatography-tandem mass spectrometry method. In the present study, we explored the possibility to utilize these biomarkers for determining the oxidative damage in liver mammalian cells in vitro. Two different treatments were chosen for inducing oxidative stress in Chinese Hamster ovary cells: menadione and copper plus hydrogen peroxide (Cu2+/H2O2). Cells were incubated with the model compounds in the presence or absence of vitamin E and C, and cytotoxicity was evaluated by a nuclear-dye method. Results were compared to two fluorescent probes, H2DCF-DA and C11 -BODIPY581/591, which have been used for determining the formation of free radicals in the cells. From ten LPO degradation products, eight were increased significantly following incubation with menadione in cell lysate or incubation media. Menadione-induced oxidative stress was also confirmed by oxidation of fluorescent probes. However, no increased formation of protein oxidation products was observed. Vitamin E and C did not diminish the formation of LPO degradation products that were increased by menadione. Although Cu2+/H2O2 did not induce oxidation of fluorescent probes, it induced formation of six out of ten LPO degradation products. Vitamin E and C did not diminish the formation of LPO degradation products; vitamin C even substantially increased the formation of acetaldehyde and propanal, which is in line with its reported prooxidant action under certain conditions. Vitamin C also caused two-fold increase in Cu2+/H2O2-induced o,o'-dityrosine formation when applied simultaneously. In conclusion, our present results show that the LPO biomarker set can be used for evaluation of oxidant capacity and the toxic potential of various chemicals in an in vitro cell model. These biomarkers might even be more sensitive than measuring protein oxidation products or oxidation of fluorescent probes.

Animals↗