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PubMed2002

Use of online-dual-column extraction in conjunction with chiral liquid chromatography tandem mass spectrometry for determination of terbutaline enantiomers in human plasma.

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a beta2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0-200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94-106%.

Chromatography, Liquid↗
PubMed2002

Premotor circuits controlling eyelid movements in conjunction with vertical saccades in the cat: I. The rostral interstitial nucleus of the medial longitudinal fasciculus.

Saccadic eye movements in the vertical plane are controlled by the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) and the interstitial nucleus of Cajal. Eye movements in the vertical direction are accompanied by concurrent upper eye lid movements. These gaze-related lid movements are produced by the levator palpebrae superioris muscle, whose motoneurons are located in the caudal central subdivision (CCS) of the oculomotor nucleus. The neural circuits that direct such gaze-related lid movements were examined by use of both conventional and dual neuronal tracing methods in the cat. Injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the area of the CCS revealed a distinctive subset of retrogradely labeled neurons located in the caudomedial portion of the riMLF. This subset of riMLF neurons was not labeled when injections were localized within the oculomotor nucleus proper, without involving the CCS. Injections of biotinylated dextran amine (BDA) that included this caudomedial riMLF region anterogradely labeled axons that projected profusely throughout the CCS. Labeled terminals were seen in close association with retrogradely labeled levator palpebrae motoneurons, which were primarily found contralateral to WGA-HRP muscle injections. Ultrastructural examination revealed that most BDA-labeled terminals contained clear spherical vesicles and formed asymmetrical synaptic contacts, primarily on the proximal dendrites of WGA-HRP-labeled motoneurons. A few had pleiomorphic vesicles. In summary, these results strongly suggest that the caudomedial part of the cat riMLF is a premotor center that monosynaptically controls lid movements in conjunction with vertical saccades.

Animals↗
PubMed2007

Premotor circuits controlling eyelid movements in conjunction with vertical saccades in the cat: II. interstitial nucleus of Cajal.

Vertical saccadic eye movements are accompanied by concurrent eyelid movements in the same direction. The interstitial nucleus of Cajal (InC) controls eye position for vertical eye movements and may also control saccade-related lid position as well. This study investigates whether the InC serves as a premotor center for eyelid saccades, by employing dual-tracer methods in cats to label both the projections of the InC and the motoneurons supplying the levator palpebrae superioris (LPS) muscle, which lie in the caudal central subdivision (CCS) of the oculomotor complex. Injections of biotinylated dextran amine (BDA) into the InC anterogradely labeled axons that terminated bilaterally throughout the CCS and in the oculomotor nuclei proper. Labeled terminals lay in close association with labeled LPS motoneurons, which were retrogradely labeled following injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the muscle. Ultrastructural investigation revealed that most terminals contained spherical vesicles and formed asymmetric synaptic contacts with the labeled motoneurons. These results strongly suggest that the InC monosynaptically controls lid movements in conjunction with vertical eye movements, including saccades. To identify the neurons of origin for this pathway, WGA-HRP injections were centered in the CCS. These experiments indicate that lid and eye motoneurons may share a common source of bilateral InC input. Thus, a common vertical position signal may be employed to maintain the lid and eye at appropriate elevations during fixation, such that the lid sits just above the pupil, allowing unobstructed vision, but at the ready to protect the cornea.

Animals↗
PubMedDate not supplied

The characterization of snake venoms using capillary electrophoresis in conjunction with electrospray mass spectrometry: Black Mambas.

Capillary electrophoresis has been used in conjunction with electrospray mass spectrometry using both full-scan and selected ion monitoring modes to supply as much information as possible about the venom of Dendroaspis polylepis polylepis (Black Mamba). As an example of the application of capillary electrophoresis/electrospray mass spectrometry (CE/ESI/MS) to the analysis of a complex mixture of small proteins, we have analyzed the venom of Dendroaspis polylepis polylepis using the combined techniques. Both full-scan and selected ion monitoring modes were employed. CE/ESI/MS provides a rapid and extremely sensitive method for molecular weight determination, particularly when selected ion monitoring is employed. It has been utilized to provide sequence confirmation for those toxins which have already been described in the literature. Our methodology indicates the presence of at least 70 peptides in the molecular weight range 6000-9000.

Elapid Venoms↗
PubMed2002

Analysis of benzo[a]pyrene diol epoxide-DNA adducts by capillary zone electrophoresis- electrospray ionization-mass spectrometry in conjunction with sample stacking.

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

Hippocampal damage equally impairs memory for single items and memory for conjunctions.

In a prior study of continuous recognition performance, data were reported in support of the hypothesis that the hippocampus is not needed to remember the individual components of a stimulus but is important for remembering associations between its components (Kroll et al. 1996. J Mem Lang 35:176-196). Patients with left hippocampal damage were able to endorse recently encountered words and to reject novel words, as well as disyllabic words in which one of the syllables had been previously encountered. However, they failed to reject words in which both syllables had been encountered independently in different words. We present data from five experiments designed to examine this finding in more detail. In each experiment, five patients with bilateral hippocampal damage and eight controls were tested using the same protocol as Kroll et al. (1996). On each trial, a two-component stimulus was presented. Stimuli could be entirely novel, novel with one previously encountered (repeated) component, novel but with both components repeated, or a true repetition. The first experiment was a direct replication using the same disyllabic words as Kroll et al. (1996). The second experiment used pseudo-words, constructed of two monosyllabic words (e.g., jambark). The third experiment used the same pairs of monosyllabic words, but presented separately on the screen to encourage participants to treat each component independently. The fourth experiment used pairs of objects, and the fifth experiment used face-house pairs. In all five experiments, patients with hippocampal damage exhibited impaired recognition memory. The impairment extended across all trial types with no evidence that hippocampal damage selectively (or disproportionately) impaired the associative or conjunctive component of memory. We discuss our findings in the light of the work by Kroll et al. (1996) and other recent neuropsychological, electrophysiological, and neuroimaging studies of hippocampal function and single-item and associative memory.

Adult↗
PubMed1982

Antitumor effect of arginine butyrate in conjunction with Corynebacterium parvum and interferon.

The antitumor effect of arginine butyrate, used alone or in conjunction with interferon (IFN), was studied using randomly selected Swiss mice inoculated i.p. with 10(6) 180 TG Crocker tumor cells. The results of these different therapeutic regimens were estimated by tumor incidence at 10 days, by the time necessary to protect 50% of the animals, and by the final survival rate. Combined treatment by IFN and arginine butyrate was effective; but in order to compensate for IFN's immune repressive effects, a single injection of Corynebacterium parvum was administered in some experiments prior to IFN and/or arginine butyrate. After this immune potentiation, both IFN and arginine butyrate used separately significantly protected the animals. However, optimal results were obtained when a single C. parvum injection was followed by nine daily alternating shots of arginine butyrate and IFN.

Animals↗
PubMed1990

CBP7 codes for a co-factor required in conjunction with a mitochondrial maturase for splicing of its cognate intervening sequence.

Maturases are required for the processing of their cognate intervening sequences in the mitochondrial cytochrome beta pre-mRNA. In this paper we characterize a nuclear gene, CBP7, already known to be required for the translation of the cytochrome b transcript; we present further evidence that it is also required as a co-factor in conjunction with the maturases for the efficient processing of their intervening sequences. Results are based on Northern blot analyses which show that in cbp7 mutants with the short cytochrome b gene the maturase encoding intervening sequence fails to be excised although, at the same time, at least wild-type levels of the maturase are present as indicated by antibodies specific to it in Western blot analyses.

Cytochrome b Group↗
PubMed1992

Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3-3'-diaminobenzidine and immunocytochemistry.

We describe the application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver. For automatic detection by light and electron microscopy peroxisomes must be stained with the alkaline DAB procedure for catalase. There is a good agreement between the results obtained by conventional morphometric techniques and by automatic image analysis of DAB-stained electron microscopic preparations. Moreover, the image analyzer may be used in conjunction with a light microscope for evaluation of semithin sections (1-0.25 microns), provided the section thickness factor is taken into consideration. This latter approach has proven highly efficient in estimation of peroxisome proliferation. The limitations of this method and the relevance of volume density as a reliable morphometric parameter for evaluation of peroxisome proliferation are discussed. In the second part of this study we present the application of image analysis for quantitation of alterations of individual peroxisomal enzyme proteins after treatment with bezafibrate in immunogold stained ultrathin sections. There is good agreement between the results of quantitative immunocytochemistry and Western (immuno) blot analysis of highly purified peroxisomal fractions. In our experience quantitative immunoelectron microscopy provides a versatile, highly sensitive, and efficient method for detection of modulations of various proteins in peroxisomes. Finally the limitations and prospects of quantitative immunocytochemistry for investigation of peroxisomal proteins are discussed.

3,3'-Diaminobenzidine↗
PubMed2005

Short breath-hold, volumetric coronary MR angiography employing steady-state free precession in conjunction with parallel imaging.

An ECG-gated, 3D steady-state free precession (SSFP) technique in conjunction with sensitivity encoding (SENSE)-based parallel imaging was implemented for short breath-hold, volumetric coronary MR angiograpy (CMRA). Two parallel imaging acquisition strategies (employing 1 R-R and 2 R-R intervals, respectively) were developed to achieve 1) very short breath-hold times (12 s for a heart rate of 60 bpm), and 2) small acquisition windows to minimize sensitivity to physiologic motion. Both strategies were examined in CMRA applications over a range of heart rates. A four-point scale blinded reading (with 4 indicating the most desirable features) revealed substantial image quality improvements for the accelerated data as compared to the nonaccelerated approach. The 1 R-R interval scheme yielded an image score of 3.39 +/- 0.60, and was found to be particularly suitable for low heart rates (P = 0.0008). The 2 R-R interval strategy yielded an image score of 3.35 +/- 0.64, and was more appropriate for higher heart rates (P = 0.03). The results demonstrate that 3D SSFP combined with parallel imaging is a versatile method for short breath-hold CMRA while maintaining high spatial resolution. This strategy permits imaging of the major coronary artery distributions in two to three breath-holds using targeted slabs, and offers the potential for single breath-hold, large-volume CMRA.

Adult↗
PubMed2006

Toxicity profile of delayed high dose sodium thiosulfate in children treated with carboplatin in conjunction with blood-brain-barrier disruption.

PURPOSE: To assess the safety of delayed high dose intravenous (i.v.) sodium thiosulfate (STS) in a case series of 12 children with malignant brain tumors who were treated with intraarterial (i.a.) carboplatin in conjunction with blood-brain-barrier disruption (BBBD). METHODS: Twelve children ages 17 months-12 years underwent a total of 132 BBBD chemotherapy treatments and also received delayed high dose STS (i.v.). Dose 1 of STS (10-16 g/m(2)) was administered 2 or 4 hr after carboplatin, and a second STS dose was administered 4 hr after dose 1 if the child had impaired baseline hearing. Toxicity data were graded in accordance with the National Cancer Institute Common Toxicity Criteria (Version 2). Audiologic monitoring to evaluate the otoprotective potential of STS was performed on 11 children. Ototoxicity was defined in accordance with the American Speech-Language-Hearing Association (ASHA) criteria. Baseline and end of treatment hearing status were graded using Brock's criteria. RESULTS: Nausea and vomiting were well controlled with anti-emetics administered approximately 30 min prior to STS infusion. Analogous to results in adult patients, there was mild transient hypernatremia and a trend for improved protection from ototoxicity in children who received STS delayed to 4 hr post-treatment versus 2 hr. Tumor responses were seen in heavily pre-treated patients with relatively chemo-resistant tumors, suggesting that STS did not protect the tumor from platinum cytotoxicity. CONCLUSION: High dose STS is well tolerated in children under 12 years of age. Further studies of STS in children are warranted to assess otoprotection and the impact of STS on platinum mediated efficacy.

Antineoplastic Agents↗
PubMed2004

Solution conformational study of nociceptin an(d its 1-13 and 1-11 fragments using circular dichroism and two-dimensional NMR in conjunction with theoretical conformational analysis.

Conformational studies of nociceptin (NC-NH2), its fully active fragment, NC(1-13)-NH2, and two significantly less potent fragments, NC(1-13)-OH and NC(1-11)-OH, were conducted in water and TFE solutions by the employment of circular dichroism, and in DMSO-d6 by 2DNMR spectroscopy in conjunction with theoretical conformational analysis. The conformations of all thepeptides studied were calculated taking two approaches. The first assumes multiconformational equilibrium of the peptide studied, which is characterized by a set of conformations (and their statistical weight values)obtained from a global conformational analysis using three methods: the electrostatically driven Monte-Carlo (EDMC) with the ECEPP/3 force field, the simulated annealing (SA) protocols in the AMBER and CHARMM force fields. The second approach incorporates the interproton distance and dihedral angle constraints into the starting conformation. Calculations were performed using the distance geometry and SA protocol in the CHARMM force field implemented in the X-PLOR program. The CD experiments indicated that for the active peptides, hydrophobic solvents induced a significantly higher (compared with those remaining)content order, probably a helical structure. Unfortunately, as a result of the conformational flexibility of thepeptides, the analysis of conformations obtained with both approaches and different force fields did not alllow the selection of any structural elements of the NC peptides that might be connected with their bioactivity. The only common element found in most conformations of the active peptides was a helical character of fragment 8-13, which allowed the side chains of basic amino acid residues to be exposed to the outside of the molecule and probably to interact with the ORL1 receptor.

Animals↗
PubMed2002

India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.

Amino Acid Sequence↗
PubMed1993

The measurement of urinary beta core fragment in conjunction with serum CA125 does not aid the differentiation of malignant from benign pelvic masses.

The urinary concentration of the renal metabolite of the beta subunit of human chorionic gonadotropin (beta core) has been proposed as a tumor marker in certain nontrophoblastic malignancies including those of the female genital tract. A previous study investigated the use of urinary beta core in conjunction with serum CA125 in distinguishing malignant from benign pelvic masses and showed that the combined test improved the overall sensitivity to 88%; this was greater than that for either test alone. However, the cutoff levels used to distinguish normal from abnormal were approximately six times greater than those generally used for CA125 and four to five times lower than those used for beta core in our laboratory. Furthermore there was no recognition of the possible difference in normal levels of beta core between pre- and postmenopausal women. We have examined a similar group of cases using our cutoff levels for urinary beta core of 0.36 ng/ml in premenopausal women and 0.48 ng/ml in postmenopausal women and 35 u/ml for CA125. We show that measurement of CA125 is substantially more sensitive that that of beta core and that the combination of beta core with CA125 does not improve the overall sensitivity of the test. However, there was a small improvement in positive predictive value if both tests were positive (97.5%) and of specificity when one or the other test was negative (98.5%).

Adult↗
PubMed1998

Use of differential display in conjunction with bulked segregants to target specific genomic loci.

Differential display has proven to be a very successful technique for isolating differentially expressed transcripts. We sought to expand the capabilities of the technique by attempting to isolate cDNAs from specific genomic loci. Two loci of interest to us are the S locus and the MOD locus, both involved in the self-incompatibility phenomenon of Brassica. The S locus is a complex locus for which molecular markers have previously been isolated, and the MOD locus is a single-gene locus for which no markers are available. We used plant material from F2 populations that segregate for two allelic variants of each locus to create two bulks or pools of plants for each differential display screen. Pooling F2 individuals effectively homogenizes background polymorphisms found in the parent plants. RNA was prepared from each bulk and differential display was performed using a kit from GenHunter Corporation (Nashville, TN). One cDNA that segregated completely with the target locus was isolated from each screen. Multiple cDNAs that were linked to each locus were also identified. We have demonstrated that differential display, when used in conjunction with bulked segregants, is a very powerful technique for isolating cDNAs from genomic loci of interest.

Brassica↗
PubMed2001

Mapping motor inhibition: conjunctive brain activations across different versions of go/no-go and stop tasks.

Conjunction analysis methods were used in functional magnetic resonance imaging to investigate brain regions commonly activated in subjects performing different versions of go/no-go and stop tasks, differing in probability of inhibitory signals and/or contrast conditions. Generic brain activation maps highlighted brain regions commonly activated in (a) two different go/no-go task versions, (b) three different stop task versions, and (c) all 5 inhibition task versions. Comparison between the generic activation maps of stop and go/no-go task versions revealed inhibitory mechanisms specific to go/no-go or stop task performance in 15 healthy, right-handed, male adults. In the go/no-go task a motor response had to be selectively executed or inhibited in either 50% or 30% of trials. In the stop task, the motor response to a go-stimulus had to be retracted on either 50 or 30% of trials, indicated by a stop signal, shortly (250 ms) following the go-stimulus. The shared "inhibitory" neurocognitive network by all inhibition tasks comprised mesial, medial, and inferior frontal and parietal cortices. Generic activation of the go/no-go task versions identified bilateral, but more predominantly left hemispheric mesial, medial, and inferior frontal and parietal cortices. Common activation to all stop task versions was in predominantly right hemispheric anterior cingulate, supplementary motor area, inferior prefrontal, and parietal cortices. On direct comparison between generic stop and go/no-go activation maps increased BOLD signal was observed in left hemispheric dorsolateral prefrontal, medial, and parietal cortices during the go/no-go task, presumably reflecting a left frontoparietal specialization for response selection.

Adult↗
PubMed1987

Chemotherapy administered in conjunction with osmotic blood-brain barrier modification in patients with brain metastases.

Clinical experience in seven patients with systemic malignancies (breast, lung and testicular) metastatic to the central nervous system treated with chemotherapy given in association with reversible osmotic blood-brain barrier modification is reviewed. A combination chemotherapy regimen including intra-arterial methotrexate, intravenous cytoxan and oral procarbazine in conjunction with intra-arterial mannitol infusions was successfully carried out with minimal toxicity. The results in these patients demonstrate some therapeutic efficacy to the increased drug delivery achieved with this technique. Although suggestive, additional studies will be required to confirm that barrier modification is a key parameter in such efficacy.

Adult↗