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PubMed2004

Propylene glycol increases cytosolic free calcium in rat cerebrocortical synaptosomes.

In these studies, the authors investigated the effect of propylene glycol (PG) on the cytosolic free Ca2+ concentration ([Ca2+]i) in rat cerebrocortical synaptosomes using the fluorescent Ca2+ indicator fura-2. PG (0.5-5% v/v) increased [Ca2+]i in a concentration-dependent manner. The PG-induced increase in [Ca2+]i was inhibited approximately 50% by the omission of extracellular Ca2+ or the addition of Ni2+ (100 microM). Decrease of extracellular Na+ (6.2 mM) or addition of tetrodotoxin (1 microM), verapamil (10 microM), nifedipine (10 microM), omega-agatoxin IVA (200 nM), omega-conotoxin GVIA (1 microM), or omega-conotoxin MVIIC (1 microM) had no effect on the increase in [Ca2+]i. Also, addition of TMB-8 (100 microM), ryanodine (50 microM) or thapsigargin (1 microM) did not modify the increase in [Ca2+]i in the absence of extracellular Ca2+. These results suggest that PG increases [Ca2+]i in rat cerebrocortical synaptosomes by both stimulating Ca2+ entry through a Ni2+-sensitive pathway and releasing Ca2+ from TMB-8-, ryanodine- and thapsigargin-insensitive Ca2+ stores.

Animals↗
PubMed2000

Comparison of oxime-initiated reactivation of organophosphorous-inhibited acetylcholinesterase in brains of avian embryos.

Organophosphorous (OP) insecticide-induced inhibition and oxime reactivation of acetylcholinesterase (AChE) was determined in whole-brain homogenates prepared from 15-d-old chick embryos. Doses of chlorpyrifos, parathion, acephate, and trichlorfon that inhibited AChE >70% were administered to the embryos. Following insecticide exposure, an in vitro system compared the capability of the oximes pralidoxime (2-PAM), obidoxime, TMB-4, and HI-6 to reactivate the OP-inhibited AChE. Concentration-related increases in AChE activities were noted in embryo brains reactivated with 2-PAM, TMB, and HI-6. 2-PAM was the most effective reactivator of trichlorfon-inhibited AChE; 2-PAM and obidoxime were relatively similar in effectiveness for reactivation of AChE inhibited with the other OP insecticides used as test agents. All oximes were similarly effective against acephate, but HI-6 was the least effective reactivator of AChE in chick embryo brain homogenates inhibited by the other OP insecticides. These results suggest that both the OP insecticide inhibiting AChE and the oxime reactivating this enzyme can contribute to the effectiveness of the avian brain AChE reactivation.

Acetylcholinesterase↗
PubMed1990

Differential effects of Ca2+ on proliferation of stomach, colonic, and pancreatic cancer cell lines in vitro.

Calcium intake inhibits growth of colon cancer in vivo, the mechanisms of which are not fully elucidated. The objective of this study was to determine whether Ca2+ directly affects the growth of colon cancer cells in vitro and to compare the effects of Ca2+ on the growth of several gastroenteropancreatic cancer cells, including mouse colon cancer (MC-26), human colon cancer (LoVo and WIDR), human gastric cancer (AGS and SII), and human pancreatic cancer (PANC-1 and MIA) cells. All tumor cell lines tested grew in medium containing low concentration (approx 0.16 mM) of Ca2+. Higher concentrations of Ca2+ significantly inhibited the growth of all three colon cancer cell lines tested but had no significant effect on proliferation of the stomach and pancreatic cancer cell lines. Growth of AGS cells, in the presence of 0.1 or 0.5 mM EGTA (resulting in the loss of the extracellular Ca2+) was similar to that observed in the absence of EGTA, indicating that AGS cells were relatively insensitive to loss of extracellular Ca2+. In the presence of TMB-8, an inhibitor of intracellular Ca2+ release, the growth of colonic cancer cell lines was inhibited in a dose-dependent manner, indicating that a minimum basal level of intracellular Ca2+ was required for continued proliferation of colon cancer cells. The stomach cancer cell lines (AGS) was once again less sensitive to the effects of TMB-8 than were the colon cancer cells, indicating an inherent difference in Ca2+ requirements and sensitivity to Ca2+ for growth of different gastroenteropancreatic cancer cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Effect of signal transduction inhibition in adapted lymphocytes: micronuclei frequency and DNA repair.

Irradiation of human lymphocytes (1 cGy X rays, 37 degrees C) or their treatment with 10 microM hydrogen peroxide (30 min at 37 degrees C) evoked a ca 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). The response was reflected in a lower micronuclei frequency, but no change in DNA repair rate was observed as measured by the comet assay, directly after the challenge dose. Treatment of lymphocytes with staurosporine, an inhibitor of protein kinases, or with TMB-8, a calcium antagonist, carried out in parallel with the adaptive dose prevented the development of the adaptive response measured as micronuclei frequency. In lymphocytes that were staurosporine- or TMB-8-treated and irradiated under adaptive conditions showed that the rate of DNA repair was not changed. We conclude that treatment with agents that interfere with the transduction of the signal triggered by the low dose prevents the development of the adaptive response induced by X rays or hydrogen peroxide. Lower chromosome damage revealed by the cytokinesis block-micronuclei test in the adapted lymphocytes is unrelated to DNA repair rate as measured by comet assay.

Adaptation, Physiological↗
PubMed2005

Effect of in vivo jet fuel exposure on subsequent in vitro dermal absorption of individual aromatic and aliphatic hydrocarbon fuel constituents.

The percutaneous absorption of topically applied jet fuel hydrocarbons (HC) through skin previously exposed to jet fuel has not been investigated, although this exposure scenario is the occupational norm. Pigs were exposed to JP-8 jet fuel-soaked cotton fabrics for 1 and 4 d with repeated daily exposures. Preexposed and unexposed skin was then dermatomed and placed in flow-through in vitro diffusion cells. Five cells with exposed skin and four cells with unexposed skin were dosed with a mixture of 14 different HC consisting of nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, ethyl benzene, o-xylene, trimethyl benzene (TMB), cyclohexyl benzene (CHB), naphthalene, and dimethyl naphthalene (DMN) in water + ethanol (50:50) as diluent. Another five cells containing only JP-8-exposed skin were dosed solely with diluent in order to determine the skin retention of jet fuel HC. The absorption parameters of flux, diffusivity, and permeability were calculated for the studied HC. The data indicated that there was a two-fold and four-fold increase in absorption of specific aromatic HC like ethyl benzene, o-xylene, and TMB through 1- and 4-dJP-8 preexposed skin, respectively. Similarly, dodecane and tridecane were absorbed more in 4-d than 1-dJP-8 preexposed skin experiments. The absorption of naphthalene and DMN was 1.5 times greater than the controls in both 1- and 4-d preexposures. CHB, naphthalene, and DMN had significant persistent skin retention in 4-d preexposures as compared to 1-d exposures that might leave skin capable of further absorption several days postexposure. The possible mechanism of an increase in HC absorption in fuel preexposed skin may be via lipid extraction from the stratum corneum as indicated by Fourier transform infrared (FTIR) spectroscopy. This study suggests that the preexposure of skin to jet fuel enhances the subsequent in vitro percutaneous absorption of HC, so single-dose absorption data for jet fuel HC from naive skin may not be optimal to predict the toxic potential for repeated exposures. For certain compounds, persistent absorption may occur days after the initial exposure.

Animals↗
PubMed1995

Use of progesterone-3(O-carboxymethyl oxime)-horseradish peroxidase in a sensitive microtitre-plate EIA and its application to a visual membrane EIA of progesterone.

A simple method of visual membrane enzyme-immunoassay (EIA) for the detection of progesterone is described. When two types of progesterone-horseradish peroxidase (HRP) tracers were challenged for binding, in the presence of progesterone, to the monoclonal anti-progesterone antibody, 15A, coated on the microtitre plate, the HRP conjugated at the C-3 position (A-ring) of progesterone competed more effectively with progesterone to the binding site of the monoclonal antibody (mAb) than HRP conjugated at the C-11 position of the C-ring. By using this combination of mAb, 15A, and progesterone-3(O-carboxymethyloxime)-HRP (P-3CMO-HRP), we developed a visual membrane EIA system in which free progesterone in the sample could be quantified by the degree of color development. In this system, free progesterone competed with P-3CMO-HRP for binding sites of mAb immobilized on the nitrocellulose membrane. The stable grey color was formed on the surface of membrane for progesterone-negative and no color for progesterone-positive sample using 3,3'-diaminobenzidine (DAB) with Co2+ as an insoluble substrate solution. To examine whether tetramethylbenzidine (TMB) can substitute for DAB in membrane EIA, an experiment was conducted where TMB was used as an insoluble substrate.

3,3'-Diaminobenzidine↗
PubMed2004

Conformational control of Bax localization and apoptotic activity by Pro168.

In healthy cells, Bax resides inactive in the cytosol because its COOH-terminal transmembrane region (TMB) is tucked into a hydrophobic pocket. During apoptosis, Bax undergoes a conformational change involving NH2-terminal exposure and translocates to mitochondria to release apoptogenic factors. How this process is regulated remains unknown. We show that the TMB of Bax is both necessary and sufficient for mitochondrial targeting. However, its availability for targeting depends on Pro168 located within the preceding loop region. Pro168 mutants of Bax lack apoptotic activity, cannot rescue the apoptosis-resistant phenotype of Bax/Bak double knockout cells, and are retained in the cytosol even in response to apoptotic stimuli. Moreover, the mutants have their NH2 termini exposed. We propose that Pro168 links the NH2 and the COOH terminus of Bax and is required for COOH-terminal release and mitochondrial targeting once this link is broken.

Amino Acid Sequence↗
PubMed1998

A Ca2+-induced Ca2+ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals.

The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20-50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05-0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N, N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN-. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.

Action Potentials↗
PubMed1995

Torsional bond strength and failure pattern of ceramic brackets bonded to composite resin laminate veneers.

The objectives of this study were to investigate the torsional bond strength of ceramic brackets bonded to composite resin veneer laminates and to human enamel. Microfilled resin veneers were bonded directly to prepared, etched bovine teeth embedded in epoxy resin. Brackets [Allure IV (NSB), Fascination, Starfire TMB, and Transcend 2000] were bonded to abraded, acid-etched resin veneers with a light-cured or a chemically-cured adhesive. Brackets were also bonded to human teeth with light-cured and chemically-cured adhesives for comparison purposes. After 24 hours storage in water, specimens were subjected to torsional stress and the maximum shear stress tau max, was calculated. The debonded brackets, the veneer, and enamel surfaces were examined under a stereomicroscope and a SEM to study the failure modes. Three-way ANOVA with a Tukey multiple comparisons test revealed significant differences in bond strengths among bracket types and bonding substrate at P = 0.05 level. Highest bond strength was observed in brackets with a combination of micromechanical retention and chemical adhesion. Significant interactions among bracket, substrate (enamel or resin) and mode of cure of adhesive were observed. Analysis of the failure pattern of brackets revealed adhesive and/or cohesive resin failures in all brackets studied, while cohesive bracket failures occurred in the single-crystal Starfire TMB bracket and the polycrystalline Transcend 2000 bracket. Debonding ceramic brackets under a steady torsional load caused no substrate surface alterations regardless of adhesive used.

Analysis of Variance↗
PubMed1985

Effect of antagonists of calcium and phospholipase A on the cytopathogenicity of Entamoeba histolytica.

The in vitro mechanisms by which Entamoeba histolytica trophozoites lyse target Chinese hamster ovary (CHO) cells were examined. Calcium chelators ethylenediaminetetraacetate and ethyleneglycol bis (beta-aminoethyl ether)-N,N'-tetraacetate (10 mM) inhibited amebic cytolysis of target CHO cells (P less than .01). A putative antagonist of intracellular calcium flux, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8; greater than or equal to 250 microM), inhibited amebic adherence and cytolysis (P less than .001). Quinacrine, Rosenthal's inhibitor (dimethyl-dl-2,3-distearoyloxypropyl-2'-hydroxyethyl ammonium acetate), phosphatidylcholine, and hydrocortisone (greater than or equal to 10(-4) M), all pharmacological antagonists of eukaryotic phospholipase A enzymes, inhibited amebic killing of target CHO cells (P less than .001). At 37 C quinacrine and hydrocortisone reduced amebic adherence to CHO cells, whereas Rosenthal's inhibitor and phosphatidylcholine did not. Phosphatidylcholine and TMB-8 demonstrated a synergistic inhibitory effect on amebic killing of target CHO cells (P less than .001). These studies indicate that extracellular calcium ions, amebic intracellular calcium flux, and amebic phospholipase A activity are required for cytolysis of target cells by E. histolytica.

Adhesiveness↗
PubMed2002

Tripartite molecular beacons.

Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many solution-based nucleic acid detections, MBs are expensive probes for applications that require the use of a large number of different DNA probes due to the high cost and tedious procedures associated with probe synthesis and purification. In addition, since both ends of MB probes are covalently modified with chromophores, they do not offer the flexibility for fluorophore change and the capability for surface immobilization through free DNA ends. In this report, we describe an alternative form of MB, denoted tripartite molecular beacon (TMB), that may help overcome these problems. A TMB uses an unmodified oligodeoxyribonucleotide that forms a MB-like structure with two universal single-stranded arms to bring on a universal pair of oligodeoxyribonucleotides modified separately with a fluorophore and a quencher. We found that TMBs are as effective as standard MBs in signaling the presence of matching nucleic acid targets and in precisely discriminating targets that differ by a single nucleotide. TMBs have the necessary flexibility that may make MBs more affordable for various nucleic acid detection applications.

Base Sequence↗
PubMed1996

Role of calcium in tumor necrosis factor-alpha production by activated macrophages.

The role of calcium in the production of tumor necrosis factor-alpha (TNF-alpha) by the lipopolysaccharide (LPS)-stimulated macrophage cell line, J774.1, was investigated. Flow cytometric measurement of intracellular calcium concentration ([Ca]i) using 2-(3,6-bis(acetyl-oxy)-2,7-dichloro-9H-xanthen-9-yl)benzoic acid (fluo-3)-loaded J774.1 cells revealed that LPS at more than 0.1 microgram/ml increased [Ca]i transiently in the presence or absence of serum, and that a mixture of a calcium chelator, ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and a calcium release blocker from intracellular store sites, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), inhibited the [Ca]i response induced by LPS. In concordance with this, production of TNF-alpha was inhibited by EGTA and/or TMB-8. These reagents also reduced the level of TNF-alpha mRNA significantly. These results indicate that the transient increase of [Ca]i plays a role in LPS-induced expression of TNF-alpha by the macrophage cell line.

Animals↗
PubMed1987

ATP-induced calcium transient in cultured rat aortic smooth muscle cells.

To characterize the excitatory purinoceptors in vascular smooth muscle cells and the biochemical mechanisms of their actions, the effects of ATP and other nucleotides on Ca2+ mobilization in cultured smooth muscle cells mainly from rat aorta were investigated. ATP induced a transient and dose-dependent increase in the cytosolic Ca2+ concentration. ATP also induced a rapid production of inositol trisphosphate (IP3). The agonist form of ATP was metal-free ATP and its half-maximal effect was obtained at about 0.1 microM. 4-beta-Phorbol 12-myristate 13-acetate (PMA) or 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both Ca2+ response and IP3 production. In addition, TMB-8 but not PMA, significantly decreased the amount of releasable Ca2+ presumably in the sarcoplasmic reticulum. Pertussis toxin also inhibited the Ca2+ response. Based on the dose-dependent effects of various nucleotides and adenosine on the Ca2+ response, it was concluded that the P2 subclass of purinoceptor is involved in the observed ATP effects. In addition, the observed absence or very weak effect of alpha, beta-methylene ATP relative to the effect of ATP suggests that the excitatory P2-purinoceptors in vascular smooth muscle cells do not form a homogeneous group, because the opposite order of potency for these two nucleotides was reported previously for the P2 purinoceptors involved in contraction of some isolated blood vessels.

Adenosine Triphosphate↗
PubMed2001

Moderator variables of executive functioning in schizophrenia: meta-analytic findings.

The literature regarding executive functioning in schizophrenia was examined to determine effect size differences between schizophrenic, normal control, and psychiatric groups. Effect sizes (n = 176) from 71 studies were cumulated across measures including the Wisconsin Card Sorting Test (WCST), Halstead Category Test (HCT), verbal/design fluency, Trail Making B (TMB), and the Stroop Color-Word Test. Schizophrenic groups were impaired relative to controls (delta = -1.45). Schizophrenic groups also performed 0.40 standard deviations lower than psychiatric groups. Within the schizophrenic versus normal control comparisons, the effect size based on complex measures, such as the WCST/HCT (delta = -1.42), was similar to that based on less complex measures, such as TMB and the Stroop (delta = -1.58). Chlorpromazine equivalents and illness duration were not significantly correlated with effect sizes (p > 0.05). Effect sizes and number of hospitalizations (p < 0.05) were related. Effect sizes and Brief Psychiatric Rating Scale (BPRS) were not significantly correlated. Negative and positive symptoms were significantly correlated (p < 0.05). The results as they apply to research and clinical practice are discussed.

Adult↗
PubMed2004

Use of assisted reproductive technologies in the propagation of rhesus macaque offspring.

The assisted reproductive technologies (ARTs) as tailored to the production of rhesus monkeys at the Oregon National Primate Research Center (ONPRC) are described. Efficient fertilization of mature oocytes recovered by aspiration from females subjected to follicular stimulation was achieved with fresh or frozen sperm by intracytoplasmic sperm injection (ICSI). Embryo development to the early cleavage stage occurred at high frequency. Cryopreserved embryos showed high postthaw survival and were also transferred in efforts to establish pregnancies. Three methods of transfer were evaluated, two involving embryo placement into the oviduct, laparoscopy and minilaparotomy, and a nonsurgical, transcervical approach that resulted in uterine deposition. Early cleaving embryos (Days 1-4) were transferred into the oviducts of synchronized recipients with optimal results and pregnancy rates of up to 36%. Pregnancy rates were similar when two fresh or frozen embryos were transferred (28- 30%), although more than two embryos had to be thawed to compensate for embryo loss during freeze-thawing. Normal gestational lengths, birth weights, and growth curves were seen with ART-produced infants compared with infants produced by natural mating in the timed mated breeding (TMB) colony at the ONPRC. In 72 singleton pregnancies established following the transfer of ART-produced embryos, the live-birth rate, at 87.5%, was statistically identical to that for the TMB colony. Further development of the ARTs should result in increasing use of these techniques to augment conventional approaches to propagating monkeys, especially those of defined genotypes.

Animals↗
PubMed1991

Bile acid stimulation of cyclic AMP and ion transport in developing rabbit colon.

Bile acids elicit Cl secretion and increases in short circuit current (Isc) in rabbit distal colon in vitro in adult but not newborn animals. In this investigation we found that concentrations of taurodeoxycholic acid (TDC) as low as 50 microM significantly increase cyclic AMP (cAMP) in adult but not newborn colon. Further, blocking the increases of cAMP in adult colon with 3,4,5-trimethoxybenzoate 8-(N,N-dimethylamino) octyl ester (TMB-8), partially inhibited the effect of TDC on Cl secretion. TMB-8 did not block the effect of increases in cAMP seen with vasoactive intestinal peptide (VIP), theophylline, or forskolin. When newborn colon was exposed to 1 mM TDC, limited Cl secretion was elicited. Increased cAMP is not seen in newborn colon where TDC-induced secretion is absent. Thus, increases in cAMP may represent one part of the coupling of TDC stimulation to Cl secretion.

Animals↗
PubMed1987

Double action of nicardipine on serotonin release from rat platelets.

We studied the effect of nicardipine on the release of preincorporated tritiated serotonin (5-HT) from washed rat platelets and on platelet 5-HT uptake. Nicardipine exerted a complex action. It partially antagonized thrombin-induced 5-HT secretion, but also induced the "selective" release of 5-HT without concomitant ATP release in the absence of platelet activation and inhibited active 5-HT uptake. The two latter effects were observed in the presence of EGTA and therefore could not be related to calcium channel blockade. Verapamil, TMB 8, and the calmodulin antagonists trifluoperazine and R 24571 also possessed this multiplicity of action; however, verapamil and TMB 8 were weaker inducers of selective 5-HT release and uptake inhibition. These results show that, in addition to its inhibitory effect on thrombin-induced platelet activation, nicardipine inhibits platelet 5-HT accumulation. Its administration may therefore result in 5-HT depletion.

Animals↗