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PubMedDate not supplied

Altered drug sensitivities to anticancer agents in radiation-sensitive DNA repair deficient yeast mutants.

We studied whether cellular sensitivity to anticancer agents was correlated with a repair deficiency in three yeast epistasis groups of radiation sensitive mutants. All these mutants were hypersensitive to cisplatin and mitomycin C. By contrast, both rad51 and rad52 mutants deficient in double-strand breaks repair were hypersensitive to adriamycin and bleomycin, but the rad1 and rad10 mutants deficient in nucleotide excision repair were not. These results were confirmed by examining the cellular sensitivity of either revertants or strains carrying wild RAD+ protein expression plasmids to various drugs. Cellular damage by the above anticancer agents is discussed in relation to the DNA repair mechanisms.

Antineoplastic Agents↗
PubMed1995

Effects of various cytochrome P-450 inducers on testosterone metabolism and several monooxygenase activities in Sprague-Dawley (SpD), high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats.

Hydroxylation of testosterone (TST) has been shown to be regio- and stereo-specific for a number of cytochrome P-450 isoenzymes. Three rat lines [Sprague-Dawley (SpD), high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS)] were tested for this enzymatic specificity after treatment with phenobarbital, clofibrate, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile. These compounds are known to induce cytochrome P-450 2B, 4A, 1A and 3A1, respectively, in the rat. Induction efficiency was established by using the usual enzyme activities specific for these P-450s (pentoxyresorufin, lauric acid, ethoxyresorufin and nifedipine oxidase). Five mono hydroxylated TST metabolites were separated using a sensitive HPLC procedure. The hydroxylation of TST was found to be significantly different between the lines even in the uninduced state. The formation of the metabolites of TST, hydroxylated on 2 alpha or 7 alpha or 16 alpha positions and oxidated on carbon 17 (delta 4), was found to be significantly increased in SpD rats when compared with the HAS-LAS lines (P < 0.0001 in each case). When the HAS-LAS lines were compared, the quantity of 2 alpha and 16 alpha hydroxylated metabolites was found to be significantly lower in LAS rats (P < 0.05). These differences persisted, although in the opposite direction, after 3-methylcholanthrene (P < 0.01 for both 2 alpha and 16 alpha) and phenobarbital induction (P < 0.01 for 2 alpha).(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholism↗
PubMed1995

Regulation of jejunal arterioles by capsaicin-sensitive nerves in Nippostrongylus brasiliensis-sensitized rats.

The mechanisms by which sensory nerves elicit dilation of serosal arterioles in the jejunum of rats sensitized to the nematode Nippostrongylus brasiliensis were studied using intravital microscopy. Capsaicin (0.002-2 micrograms), applied as a bolus topically to the serosa, produced a substantially larger dilation in the sensitized rats than in unsensitized rats. Abolition of the primary afferent nerves by neonatal treatment with capsaicin and blockade of capsaicin with the antagonist ruthenium red reduced markedly the dilator actions of capsaicin. Mast cell products are important in the actions of capsaicin, because pretreatment with dexamethasone (4 mg/kg), to eliminate mast cells by a macrophage-dependent mechanism, abrogated the actions of capsaicin. In addition, superfusion of the H1 receptor antagonist diphenhydramine (2 microM) blocked the actions of capsaicin. Neither cyclooxygenase products nor platelet-activating factor was involved in capsaicin-induced dilation. The actions of capsaicin and histamine were mediated via a nitric oxide (NO)-dependent mechanism, because superfusion of an inhibitor of NO synthase (NG-nitro L-arginine methyl ester, 10 microM) blocked their effects. This inhibition of capsaicin-elicited dilation by NG-nitro L-arginine methyl ester was prevented by L-arginine (100 microM), the substrate for NO synthase. Thus the arteriolar dilation evoked by capsaicin activation of primary afferent nerves in N. brasiliensis-sensitized rats involves predominantly the release from mast cells of histamine, which then dilates the vessels by a NO-dependent mechanism.

Animals↗
PubMed1994

Identification of temperature-sensitive mutants of the human immunodeficiency virus type 1 protease through saturation mutagenesis. Amino acid side chain requirements for temperature sensitivity.

Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus. An Escherichia coli expression and processing assay system was used to screen 285 protease mutants for temperature-sensitive activity. Fourteen protease mutants had a temperature-sensitive phenotype, and approximately half resulted from conservative amino acid substitutions. Of the 14 substitutions that conferred a temperature-sensitive phenotype, 11 substitutions occurred at 6 positions that represent 3 pairs of residues in the protease that contact each other in the three-dimensional structure. These mutants assist in pinpointing regions of the protease that are important for enzyme activity and stability.

Amino Acids↗
PubMed1995

Plasmodium berghei: sensitivity of chloroquine-resistant and chloroquine-sensitive strains to irradiation and the effect of irradiated malaria parasites on cytochrome P450-dependent monooxygenases.

Differences in sensitivities of chloroquine-sensitive and chloroquine-resistant strains of Plasmodium berghei were observed following irradiation of the parasites. A dose of 15 kilorads from a cobalt-60 source killed the erythrocytic stages of the chloroquine-sensitive strain and no parasitemias were observed when mice were injected with these irradiated parasites. In contrast, when the chloroquine-resistant strain was irradiated with the same dose of cobalt-60 and injected into mice, an infection rate of 12.5% was observed, indicating that the latter strain was more resistant to inactivation by irradiation. Following injection of these irradiated strains of P. berghei into mice, significant decreases in mouse hepatic cytochrome P450 and benzo(a)pyrene hydroxylase activity, with no significant effect on N-demethylase activity, were observed. Serum glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) levels of mice injected with the irradiated parasites fell within the range of the serum enzyme levels in normal laboratory mice.

Alanine Transaminase↗
PubMed1977

Soluble factors in tolerance and contact sensitivity to 2,4-dinitrofluorobenzene in mice. I. Suppression of contact sensitivity by soluble suppressor factor released in vitro by lymph node cell populations containing specific suppressor cells.

Tolerance to 2,4-dinitrofluorobenzene (DNFB) contact sensitivity is in part mediated by suppressor thymus-derived cells (T cells) which are induced by pretreatment with the hapten 2,4-dinitrobenzenesulfonate. If lymph node cell suspensions containing suppressor cells are cultured in vitro, soluble suppressor factor (SSF) is released into the supernatant. When DNFB-immune lymph node cells are incubated with SSF, their ability to transfer contact sensitivity to normal recipients is suppressed. In order for SSF to be produced and/or released, it was necessary to paint the tolerant animals with DNFB 16 to 20 hr before the lymph node cells were cultured, suggesting that SSF was made in response to antigen stimulation. Specificity studies showed that SSF was both antigen specific and strain specific in its action. In addition, it was found that SSF could be absorbed by 2,4-dinitrophenyl-keyhold limpet hemocyanin (DNP-KLH) (not by KLH alone) and by anti-H-2 antibodies but not by trinitrophenyl-KLH, anti-immunoglobulin, or anti-DNP antibodies. Taken together, these results indicate that SSF is a product of the major histocompatibility complex which, although not antibody, has affinity and specificity for the hapten DNP. Furthermore, in order for SSF to suppress DNFB sensitivity, identity is required among genes in the H-2 complex between the donor of SSF and the immune lymph node cells.

Animals↗
PubMed1998

Seizure sensitivity and GABAergic modulation of ethanol sensitivity in selectively bred FAST and SLOW mouse lines.

FAST and SLOW selected mouse lines were bred for differences in locomotor response to low-dose ethanol. FAST mice exhibit an extreme stimulant response and SLOW mice exhibit locomotor depression at the same ethanol dose. We tested the hypothesis that gamma-aminobutyric acid (GABA) systems modulate ethanol's stimulant effects by examining convulsant responses to GABAA receptor ligands, and by assessing the effects of GABAA and GABAB ligands on locomotor activity in the presence and absence of EtOH. FAST mice were more sensitive to the convulsant effects of GABAA drugs, and to one of two non-GABAergic drugs also tested. FAST and SLOW mice differed in locomotor responses to two benzodiazepines, but not to other GABAA receptor ligands. Ethanol's stimulant effects were not selectively altered by bicuculline or picrotoxin. The selected lines differed in sensitivity to the locomotor depressant effects of the GABAB agonist, baclofen. Ethanol-stimulated activity of FAST mice was inhibited by baclofen, and this effect was reversed by administration of the GABAB antagonist, CGP-35348. These GABAB receptor mediated effects were replicated in DBA/2J inbred mice that exhibit extreme sensitivity to ethanol's stimulant effects. In summary, we found moderate to strong evidence that some sites on the GABAA receptor complex were altered as a consequence of selection of FAST and SLOW mice, but found little support for GABAA mediation of EtOH-stimulated activity. In contrast, we found moderate evidence for differential alteration of GABAB receptor function; however, GABAB receptor involvement in ethanol-stimulated activity was strongly supported by results in the selected lines and an inbred strain.

Animals↗
PubMed2003

Enhanced sensitivity electrochemical assay of low-molecular-weight heparins using rotating polyion-sensitive membrane electrodes.

Use of a novel rotating polycation-sensitive polymer membrane electrode yields sensors that can serve as simple potentiometric titration endpoint detectors for the determination of three FDA approved low-molecular-weight heparin (LMWH) anticoagulant drugs (Fragmin, Normiflo, and Lovenox). The rotating electrode configuration dramatically improves the reproducibility and increases the sensitivity for LMWH determinations by protamine titration. At a rotation speed of 3000 rpm, electrodes with optimized thin (50 microm) polymer membranes doped with dinonylnaphthalene sulfonate (DNNS) respond to low levels of protamine (<2 microg mL(-1)) with good precision (+/-1 mV, N=10), when protamine is infused continuously into a Tris-buffer solution, pH 7.4. When infusing protamine (at 5 microg min(-1)) continuously into solutions containing Fragmin, a clear endpoint is obtained, with the amount of protamine required to reach this endpoint proportional to the level of Fragmin present. A detection limit of less than 0.02 U mL(-1) Fragmin can be obtained via this new method, approximately one order of magnitude lower than that previously reported based on a non-rotating polycation electrode. Similar low detection limits can be achieved for potentiometric titrations of Normiflo and Lovenox. Such titrations can also be carried out in undiluted plasma samples containing the various LMWH species. In this case, detection of the LMWHs at clinically relevant concentrations (>0.2 U mL(-1)) can be readily achieved.

Animals↗
PubMed2005

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water.

We report on recent advances of our immunoassay for the hormone progesterone in cow's milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL(-1) and a quantification limit (LOQ) of only 2.0 pg mL(-1). These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5-15 ng mL(-1). Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL(-1) depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL(-1) for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.

Animals↗
PubMed1981

Chloroquine sensitivity of Plasmodium falciparum in Ibadan, Nigeria: II. Correlation of in vitro with in vivo sensitivity.

Plasmodium falciparum malaria was treated in 82 children with 25 mg/kg chloroquine orally over three days. They were observed for 28 days during which blood films were examined periodically for malaria parasites. Asexual forms of P. falciparum, present in the blood films of all the patients before commencing treatment, disappeared rapidly and by the third day no parasites were seen in blood films from any of them. Among the patients observed for more than three days, blood films remained negative throughout the observation period. In vitro tests of sensitivity of blood samples from 10 patients showed chloroquine concentrations of 0.5 to 0.8 nmol/ml to inhibit completely maturation from ring forms to schizonts. This suggests that P. falciparum in the Ibadan area is probably still fully sensitive to chloroquine.

Child↗
PubMed1990

Spatial sensitization in the short wavelength sensitive pathways under dichoptic viewing conditions.

Spatial sensitization (Westheimer) functions were measured under conditions that isolated the short wavelength sensitive pathways. The variable diameter pedestal and the test probe were either presented to the same eye (monoptic presentation) or to different eyes (dichoptic presentation). The most significant new finding was that a dichoptically presented, small, blue pedestal caused threshold elevations of about 1-2 long units for an S cone detected probe. However, a large pedestal caused little or no change in threshold. This result contrasts with previous results using white light stimuli, which showed that steadily presented dichoptic pedestals caused little or no threshold change. Furthermore, we show there is little masking when the probe is detected by the isolated M or L cone pathways. These data thus demonstrate a binocular, size dependent interaction revealed only when S cones detect the probe.

Contrast Sensitivity↗
PubMed2005

A novel method for assessing contrast sensitivity in the beagle dog is sensitive to age and an antioxidant enriched food.

Tasks requiring visual discrimination are commonly used in assessment of canine cognitive function. However, little is known about canine visual processing, and virtually nothing is known about the effects of age on canine visual function. This study describes a novel behavioural method developed to assess one aspect of canine visual function, namely contrast sensitivity. Four age groups (young, middle aged, old, and senior) were studied. We also included a group of middle aged to old animals that had been maintained for at least 4 years on a specially formulated food containing a broad spectrum of antioxidants and mitochondrial cofactors. Performance of this group was compared with a group in the same age range maintained on a control diet. In the first phase, all animals were trained to discriminate between two high contrast shapes. In the second phase, contrast was progressively reduced by increasing the luminance of the shapes. Performance decreased as a function of age, but the differences did not achieve statistical significance, possibly because of a small sample size in the young group. All age groups were able to acquire the initial discrimination, although the two older age groups showed slower learning. Errors increased with decreasing contrast with the maximal number of errors for the 1% contrast shape. Also, all animals on the antioxidant diet learned the task and had significantly fewer errors at the high contrast compared with the animals on the control diet. The initial results suggest that contrast sensitivity deteriorates with age in the canine while form perception is largely unaffected by age.

Aging↗
PubMed1998

A method to increase the sensitive range of pressure sensitive film.

Pressure-sensitive film is frequently used in biomechanics to document intra- and extra-articular contact pressures. This often involves the contact of two surfaces of varying curvature producing non-uniform pressure distributions. The purpose of this study was to determine the feasibility of using multiple films in such experiments to yield accurate pressure and contact area data. A composite arrangement of film was dynamically loaded using cylindrical indenters of five radii. An analytical model of each indentation was constructed to provide a standard for error analysis. The study showed that several ranges of pressure sensitive film can be used simultaneously to accurately transduce contact pressures arising from loading scenarios that produce contact pressure gradients and contact pressures that involve suprathreshold loading of a given film range.

Biomechanical Phenomena↗
PubMed1991

Assessment of potential contrast sensitivity. Part I: Preoperative prediction of contrast sensitivity following intraocular lens implantation.

Preoperative contrast sensitivity functions (CSFs) were determined for 15 cataract patients using laser-generated patterns that effectively bypassed the optical components of the eye. These functions were compared with postoperative CSFs determined using traditional methods by transmission through the optics of the eye. Accurate prediction of postoperative contrast sensitivity occurred in 91% of the cases (ten of 11) for patients with preoperative visual acuity of 20/200 or better and in 67% of all the cases (ten of 15). Prediction failures occurred when the laser interferometer was not able to penetrate the cataract or when postoperative complications occurred. These results suggest that preoperative measurement of laser interferometric CSF is a useful predictor of postoperative CSF for patients with mild to moderate cataracts that can be bypassed by the laser and with better than 20/200 preoperative visual acuity. The problem of assessment when lens opacity is advanced is addressed in Part II.

Aged↗
PubMed1997

Mass spectrometric quantification of 3-chlorotyrosine in human tissues with attomole sensitivity: a sensitive and specific marker for myeloperoxidase-catalyzed chlorination at sites of inflammation.

Oxidative modification of proteins has been implicated in a variety of processes ranging from atherosclerosis to aging. Identifying the underlying oxidation pathways has proven difficult, however, due to the lack of specific markers for distinct oxidation pathways. Previous in vitro studies demonstrated that 3-chlorotyrosine is a specific product of myeloperoxidase-catalyzed oxidative damage and that the chlorinated amino acid may thus serve as an index of phagocyte-mediated tissue injury in vivo. Here we describe a highly sensitive and specific analytical method for the quantification of 3-chlorotyrosine content of tissues. The assay combines gas chromatography with stable isotope dilution mass spectrometry, and it detects attomole levels of 3-chlorotyrosine in a single determination. Furthermore, the method is highly reproducible, with inter- and intra-sample coefficients of variance of < 3%. The specificity, sensitivity, and reproducibility of 3-chlorotyrosine determination should make this method useful for exploring the role of myeloperoxidase in catalyzing oxidative reactions in vivo.

Analysis of Variance↗
PubMed2003

Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids.

Highly sensitive biosensors based on pH-sensitive field effect transistors and cholinesterases for detection of solanaceous glycoalkaloids have been developed, characterised and optimised. The main analytical characteristics of the biosensors developed have been studied under different conditions and an optimal experimental protocol for glycoalkaloids determination in model solution has been proposed. Using such a biosensor and an enzyme reversible inhibition effect, the total potato glycoalkaloids content can be determined within the range of 0.2-100 microM depending on the type of alkaloid, with lowest detection limits of 0.2 microM for alpha-chaconine, 0.5 microM for alpha-solanine and 1 microM for solanidine. The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection of glycoalkaloids in real potato samples. High reproducibility, operational and storage stability of the biosensor developed have been shown.

Biosensing Techniques↗
PubMed2006

Sensitivity and specificity improvement of an ion sensitive field effect transistors-based biosensor for potato glycoalkaloids detection.

Butyryl cholinesterase of different origin along with variations of the time of enzyme immobilization on the potentiometric transducer surface is offered to control the ion sensitive field effect transistor (ISFET)-based biosensor sensitivity. Because butyryl cholinesterase has been already used to develop the sensors for heavy metals, organophosphorus/carbamate pesticides, and steroidal glycoalkaloids analysis, the present study has been focused on the investigation and adjustment of the ISFET-based biosensor specificity exclusively to the glycoalkaloids. Utilization of ethylendiaminetetracetate (a complexon of heavy metal ions) and phosphotriesterase (a highly efficient catalyst for the hydrolysis of organophosphorus compounds) enabled the highly specific determination of glycoalkaloids at the background of lead and mercury (up to 500 microM of ions concentration) and paraoxon (up to 100 microM of pesticide concentration). The developed biosensor has been validated for glycoalkaloids detection in potato varieties cultivated in Ukraine, and the results obtained are compared to those measured by the methods of HPLC and TLC.

Alkaloids↗
PubMed2001

A homologue of N-ethylmaleimide-sensitive factor in the malaria parasite Plasmodium falciparum is exported and localized in vesicular structures in the cytoplasm of infected erythrocytes in the brefeldin A-sensitive pathway.

N-Ethylmaleimide-sensitive factor (NSF) and its homologues play a central role in vesicular trafficking in eukaryotic cells. We have identified a NSF homologue in Plasmodium falciparum (PfNSF). The reported PfNSF gene sequence (GenBank accession number CAB10575) indicated that PfNSF comprises 783 amino acids with a calculated molecular weight of 89,133. The overall identities of its gene and amino acid sequences with those of rat NSF are 50.9 and 48.8%, respectively. Reverse transcription-polymerase chain reaction analysis and Northern blotting with total P. falciparum RNA indicated expression of the PfNSF gene. Polyclonal antibodies against a conserved region of NSF specifically recognized an 89-kDa polypeptide in the parasite cells. After homogenization of the parasite cells, approximately 90% of an 89-kDa polypeptide is associated with particulate fraction, suggesting membrane-bound nature of PfNSF. PfNSF was present within both the parasite cells and the vesicular structure outside of the parasite cells. The export of PfNSF outside of the parasite cells appears to occur at the early trophozoite stage and to terminate at the merozoite stage. The export of PfNSF is inhibited by brefeldin A, with 9 microM causing 50% inhibition. Immunoelectromicroscopy indicated that intracellular PfNSF was associated with organelles such as food vacuoles and that extracellular PfNSF was associated with vesicular structures in the erythrocyte cytoplasm. These results indicate that PfNSF expressed in the malaria parasite is exported to the extracellular space and then localized in intraerythrocytic vesicles in a brefeldin A-sensitive manner. It is suggested that a vesicular transport mechanism is involved in protein export targeted to erythrocyte membranes during intraerythrocytic development of the malaria parasite.

Amino Acid Sequence↗