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PubMed1992

Importance of peripheral insulin levels for insulin-induced suppression of glucose production in depancreatized dogs.

It is generally believed that glucose production (GP) cannot be adequately suppressed in insulin-treated diabetes because the portal-peripheral insulin gradient is absent. To determine whether suppression of GP in diabetes depends on portal insulin levels, we performed 3-h glucose and specific activity clamps in moderately hyperglycemic (10 mM) depancreatized dogs, using three protocols: (a) 54 pmol.kg-1 bolus + 5.4 pmol.kg-1.min-1 portal insulin infusion (n = 7; peripheral insulin = 170 +/- 51 pM); (b) an equimolar peripheral infusion (n = 7; peripheral insulin = 294 +/- 28 pM, P < 0.001); and (c) a half-dose peripheral infusion (n = 7), which gave comparable (157 +/- 13 pM) insulinemia to that seen in protocol 1. Glucose production, use (GU) and cycling (GC) were measured using HPLC-purified 6-[3H]- and 2-[3H]glucose. Consistent with the higher peripheral insulinemia, peripheral infusion was more effective than equimolar portal infusion in increasing GU. Unexpectedly, it was also more potent in suppressing GP (73 +/- 7 vs. 55 +/- 7% suppression between 120 and 180 min, P < 0.001). At matched peripheral insulinemia (protocols 2 and 3), not only stimulation of GU, but also suppression of GP was the same (55 +/- 7 vs. 63 +/- 4%). In the diabetic dogs at 10 mM glucose, GC was threefold higher than normal but failed to decrease with insulin infusion by either route. Glycerol, alanine, FFA, and glucagon levels decreased proportionally to peripheral insulinemia. However, the decrease in glucagon was not significantly greater in protocol 2 than in 1 or 3. When we combined all protocols, we found a correlation between the decrements in glycerol and FFAs and the decrease in GP (r = 0.6, P < 0.01). In conclusion, when suprabasal insulin levels in the physiological postprandial range are provided to moderately hyperglycemic depancreatized dogs, suppression of GP appears to be more dependent on peripheral than portal insulin concentrations and may be mainly mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and by the suppressive effect of insulin on glucagon secretion. These results suggest that a portal-peripheral insulin gradient might not be necessary to effectively suppress postprandial GP in insulin-treated diabetics.

Animals↗
PubMed2005

BCR-ABL nuclear entrapment kills human CML cells: ex vivo study on 35 patients with the combination of imatinib mesylate and leptomycin B.

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) localizes to the cell cytoplasm, where it activates proliferative and antiapoptotic signaling pathways. We previously reported that the combination of the ABL kinase inhibitor imatinib mesylate (IM) and the nuclear export inhibitor leptomycin B (LMB) traps BCR-ABL inside the nucleus, triggering the death of the leukemic cells. To evaluate the efficacy of the combination of IM and LMB on human cells we collected CD34-positive cells from 6 healthy donors and myeloid progenitors from 35 patients with CML. The sequential addition of IM and LMB generated the strongest reduction in the proliferative potential of the leukemic cells, with limited toxicity to normal myeloid precursors. Furthermore, nested reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on colonies representative of each experimental condition demonstrated that the combination of IM and LMB was the most effective regimen in reducing the number of BCR-ABL-positive colonies. The efficacy of the 2-drug association was independent of the clinical characteristics of the patients. Our results indicate that strategies aimed at the nuclear entrapment of BCR-ABL efficiently kill human leukemic cells, suggesting that the clinical development of this approach could be of significant therapeutic value for newly diagnosed and IM-resistant CML patients.

Adult↗
PubMed2000

Expression of gamma-glutamylcysteine synthetase during development.

Prematurity has been associated with low glutathione (GSH) concentrations in bronchoalveolar lavage fluid as well as in leukocytes from tracheal aspirates and peripheral blood. To elucidate whether this is caused by deficient GSH synthesis, the expression and activity of gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, GCS, EC 6.3.2.2), the rate-limiting enzyme for GSH synthesis, were measured from fetal, neonatal, and adult human liver, lung, and kidney samples. The highest activity was measured in the liver, in which mRNA expression of the catalytic GCS heavy and the regulatory light subunits, as well as activity, were, on average, similar in the various stages of development. Although GCS light subunit mRNA concentrations in the lung were higher in neonates than in fetuses and adults, enzyme activities were similar. In the adult kidney, mean enzyme activity was somewhat higher than in fetal or neonatal kidney, but this may be accounted for by the variation in the small number of samples. In conclusion, GCS is expressed and active already in the second trimester and thus low GSH concentrations found in preterm neonates appear not to be explained by deficient GSH synthesis. Other factors, such as limited availability of the GSH precursor cysteine or increased GSH consumption, may account for the lower concentrations of GSH found in preterm infants.

Adult↗
PubMed1979

The role of the carbohydrate in the stabilization, processing, and packaging of the glycosylated adrenocorticotropin-endorphin common precursor in toad pituitaries.

The neurointermediate lobes of dark adapted toads, Xenopus laevis, were incubated for 30 min in [3H]arginine, [3H]arginine plus [14C]glucosamine, or [3H]glucosamine and then chased for various time periods ranging from 1--3 h. The labeled polypeptides synthesized and secreted by the lobes were analyzed by acid-urea polyacrylamide gel electrophoresis. A glycosylated ACTH-endorphin precursor (32,000 mol wt) was synthesized during the pulse and identified by immunoprecipitation by ACTH-(11--24) antiserum. During the chase, this precursor was processed to various glycopeptides and peptides, including ACTH, beta-lipotropin, and alpha-MSH, which were subsequently secreted into the medium. An immunoprecipitable ACTH-related glycoprotein (approximately 150,000 mol wt) and other nonimmunoprecipitable glycoproteins (approximately 80,000--100,000 mol wt) were also synthesized and secreted by the neurointermediate lobe. The secretion of these glycoproteins and peptides was inhibited by dopamine. The significance of glycosylation of the precursor for the biosynthesis, processing, and secretion of the ACTH, beta-lipotropin-, and MSH-related peptides was examined by using a specific inhibitor of glycosylation, tunicamycin. Tunicamycin treatment did not affect the synthesis of the 32,000 mol wt ACTH-endorphin precursor but did prevent its glycosylation. The absence of carbohydrate on the precursor resulted in its rapid intracellular degradation. Precursors that escaped degradation were processed incompletely, leading to the formation and secretion of an unglycosylated intermediate and various other abnormal peptides. The data indicate that glycosylation of the ACTH-endorphin precursor may not be involved in the processes of intracellular transport, packaging, and secretion per se but, rather, may provide specific conformational stability to the precursor as a signal for directed limited proteolysis.

Adrenocorticotropic Hormone↗
PubMed1999

Expression of Trk receptors in the developing mouse trigeminal ganglion: in vivo evidence for NT-3 activation of TrkA and TrkB in addition to TrkC.

Animals lacking neurotrophin-3 (NT-3) are born with deficits in almost all sensory ganglia. Among these, the trigeminal ganglion is missing 70% of the normal number of neurons, a deficit which develops during the major period of neurogenesis between embryonic stages (E) 10.5 and E13.5. In order to identify the mechanisms for this deficit, we used antisera specific for TrkA, TrkB, and TrkC to characterize and compare the expression patterns of each Trk receptor in trigeminal ganglia of wild type and NT-3 mutants between E10.5 and E15.5. Strikingly, TrkA, TrkB, and TrkC proteins appear to be exclusively associated with neurons, not precursors. While some neurons show limited co-expression of Trk receptors at E11.5, by E13. 5 each neuron expresses only one Trk receptor. Neuronal birth dating and cell counts show that in wild-type animals all TrkB- and TrkC-expressing neurons are generated before E11.5, while the majority of TrkA-expressing neurons are generated between E11.5 and E13.5. In mice lacking NT-3, the initial formation of the ganglion, as assessed at E10.5, is similar to that in wild-type animals. At E11.5, however, the number of TrkC-expressing neurons is dramatically reduced and the number of TrkC-immunopositive apoptotic profiles is markedly elevated. By E13.5, TrkC-expressing neurons are virtually eliminated. At E11.5, compared to wild type, the number of TrkB-expressing neurons is also reduced and the number of TrkB immunoreactive apoptotic profiles is increased. TrkA neurons are also reduced in the NT-3 mutants, but the major deficit develops between E12.5 and E13.5 when elevated numbers of TrkA-immunoreactive apoptotic profiles are detected. Normal numbers of TrkA- and TrkB-expressing neurons are seen in a TrkC-deficient mutant. Therefore, our data provide evidence that NT-3 supports the survival of TrkA-, TrkB- and TrkC-expressing neurons in the trigeminal ganglion by activating directly each of these receptors in vivo.

Animals↗
PubMed2005

Affect and the therapeutic action of psychoanalysis.

In connection with controversial IJP articles by Stern et al. and Fonagy on the interpretation of the repressed and the recovery of past memories, the author maintains that the affect that is inherent in positive transference is at the heart of therapeutic action. Points of view put forward in the controversy (based on neurobiological knowledge) are related to Freudian metapsychology, as well as to their precursors whose scope was necessarily limited by a lack of access to more recent scientific discoveries. The author demonstrates metapsychological elements of therapeutic action inherent in the intersubjective relationship, especially identification, manifested in introjection and empathy. He describes cognitive development as spontaneously blossoming from the affective nucleus, and he explains the neuroscientific bases of this step forward. The classic (interpretative) psychoanalytic method makes up the cognitive superstructure necessary for the organisation of the mind that has sprung from the affective substructure. As a primary factor in psychic change, interpretation is limited in effectiveness to pathologies arising from the verbal phase, related to explicit memories, with no effect in the pre-verbal phase where implicit memories are to be found. Interpretation--the method used to the exclusion of all others for a century--is only partial; when used in isolation it does not meet the demands of modern broad-spectrum psychoanalysis, as the clinical material presented illustrates.

Adult↗
PubMed1978

Androgen aromatization by luteinized bovine granulosa cells in tissue culture.

Luteinized bovine granulosa cells in tissue culture contained an active 19-hydroxylase aromatase enzyme system which converted exogenous androstenedione and testosterone to oestradiol-17beta; no oestrone was detected. In the absence of exogenous androgens, the cells failed to synthesize oestrogens due to a limited capacity to synthesize androgen precursor. Theca-lutein cells, present in those CL which synthesize oestrogens, may provide androgen precursor for aromatization by the granulosa-lutein cells.

Androstenedione↗
PubMed2001

Regulation of acute cortisol synthesis by cAMP-dependent protein kinase and protein kinase C in a teleost species, the rainbow trout (Oncorhynchus mykiss).

The effects of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) on acute ACTH-stimulated cortisol secretion were assessed using a specific PKA inhibitor (H-89) and a PKC activator (phorbol 12-myristate 13-acetate, PMA) in dispersed head kidney cells of rainbow trout (Oncorhynchus mykiss). To investigate the sites of action of both PKA and PKC, pregnenolone (a cortisol precursor stemmed from the rate limiting step in cortisol synthesis) and 25-OH-cholesterol (an exogenous substrate that bypasses the rate limiting step) were used as substrates, with and without ACTH stimulation. Inhibition of PKA decreased ACTH-stimulated cortisol secretion while activation of PKC had the same effect, demonstrating that PKA stimulates and PKC inhibits cortisol synthesis. Inhibition of PKA and activation of PKC had no significant effect on pregnenolone-stimulated cortisol synthesis, indicating that both PKA and PKC act upstream from the pregnenolone step. Inhibition of PKA and activation of PKC had no significant effect on basal cortisol secretion in the presence of 25-OH-cholesterol, suggesting that PKA and PKC exert their effects on the mitochondrial cholesterol translocation step. This study provided evidence for the stimulatory role of PKA and the inhibitory role of PKC in the signalling pathways leading to cortisol synthesis in teleosts.

Adrenocorticotropic Hormone↗
PubMed1992

Banting Lecture: glucose turnover. A key to understanding the pathogenesis of diabetes (indirect effects of insulin).

This article is divided into two parts. A retrospective overview summarizes some of the work that provided the framework and tools of the more recent studies. The five novel areas of research are related to the indirect effects of insulin. Regulation of plasma glucose is of central importance in health and diabetes. Understanding this precise regulation requires sensitive isotope dilution methods that can measure the rates at which glucose is produced by the liver and used by the tissues on a minute-to-minute basis. Validation studies indicated that the non-steady-state tracer method yields reasonable results when the specific activity of plasma glucose does not change abruptly. During hyperinsulinemic glucose clamps, the decrease in specific activity of glucose can be prevented by the MSTI. During exercise, the decrease of specific activity can be only in part ameliorated by step-tracer infusion. Depancreatized dogs are used extensively as a model of selective insulin deficiency, because dog stomach secretes physiological amounts of glucagon. This strategy can avoid injections of somatostatin, which can have other affects in addition to the suppression of insulin and glucagon. In human diabetes, in addition to an increase of glucose production, there is also an increase in glucose cycling in the liver. In animal models of diabetes, mild NIDDM, and in glucose intolerance, the percentage of increments of glucose cycling are much larger than those of glucose production. We hypothesize, therefore, that measurements of glucose cycling can be used as an early marker of glucose intolerance. Application of different tracer strategies and use of the depancreatized dog as a model of diabetes, we investigated the importance of the indirect effects of insulin in the pathogenesis of diabetes. 1) Because, in the treatment of IDDM, insulin is administered by the peripheral routes we compared the relative importance of hepatic and peripheral effects of insulin in regulating the rate of glucose production. Experiments were performed in depancreatized dogs that were initially maintained at moderate hyperglycemia (10 mM) with subbasal portal insulin infusion. During the experimental period, insulin was infused either peripherally or portally at 0.9 mU.kg-1.min-1. In addition, peripheral infusions were also given at 0.45 mU.kg-1.min-1. We concluded that when suprabasal insulin levels are provided to moderately hyperglycemic depancreatized dogs, the suppression of glucose production is more dependent on peripheral than portal insulin concentrations. This indirect effect of insulin may be mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and/or by suppressive effect of insulin on glucagon secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Glucose transporter gene expression in lactating bovine gastrointestinal tract.

Absorption of glucose from the intestine and probably from various portions of the gastrointestinal tract (GIT) in lactating dairy cows can have profound implications because the availability of glucose, or glucose precursors, seems to be a limiting factor for milk synthesis. This article reports on the expression of the Na+/glucose cotransporter gene (SGLT1) in mucosal tissues from rumen, omasum, abomasum, duodenum, jejunum, ileum, and cecum of lactating cows. Sodium/glucose cotransporter mRNA transcripts were detected in polyadenylated RNA of all the GIT tissues examined and in total RNA from rumen, omasum, duodenum, jejunum, ileum, and cecum. The pattern of SGLT1 mRNA distribution in these tissues was different from that of the facilitative glucose transporter GLUT5. Sodium/glucose cotransporter protein was also detected in small intestinal membranes of lactating dairy cows. Sodium-dependent glucose uptake across small intestinal brush border membrane vesicles could be resolved into linear nonsaturable (diffusion) and saturable, carrier-mediated components. The diffusion constant of glucose from jejunal membrane vesicles was 18.3+/-2.1 pmol x mM(-1) x mg protein(-1) x s(-1). The saturable component of glucose uptake had a Vmax of 27.1+/-5.8 pmol x mg protein(-1) x s(-1) and a Km of .12+/-.05 mM. Similar transport kinetics were found in the duodenum, and they were less in the ileum. These data suggest that the GIT of lactating cows is capable of active transport of glucose from the lumen across the brush border membrane of the epithelial cells. In addition, the relatively high abundance of SGLT1 mRNA in the rumen, omasum, and cecum may imply new functional and nutritional roles for these tissues in the active transport of glucose.

Animals↗
PubMed1981

Effects of butylene and propylene glycols on body composition and fatty acid synthetase in lambs.

To test whether lipogenesis is limited by lack of glucose precursors, 45 lambs (30 kg) were randomly grouped by sex and initial weight and fed ad libitum in pens of three in a growth and slaughter experiment. The diets consisted of a basal mixed chopped hay (C), hay + 4% butylene glycol (BD) and hay + 4% propylene glycol (PG). On a dry matter basis, the crude protein content and in vitro true digestibility of the hay were 9.6 and 64%, respectively. Growth and feed intake were recorded. After 13 weeks, the lambs were killed. Blood glucose concentrations, feed intake and weight gains did not differ between diets, but intake and weight gains were minimal. BD increased (P greater than .01) blood ketones. Perirenal adipose tissue fatty acid synthetase (FAS) activity averaged 2.6, 2.4 and 1.2 nmole NADPH oxidized/mg cytosol protein/min for the BD, PG and C lambs, respectively. Values for both glycol-fed groups were higher (P greater than .01) than the control value. Carcass chemical composition followed a similar pattern, with BD, PG and C carcasses containing 38.1, 39.2 and 35.8% dry matter, respectively, and the dry matter containing 36.6, 39.4 and 30.0% fat, respectively. Carcasses of the BD and PG lambs did not differ significantly from each other, but both groups had more (P greater than .05) fat and less (P greater than .05) protein and ash than did the C carcasses, suggesting that the chemical composition of lamb carcasses may be nutritionally manipulated. The essentially equal response of adipose FAS activity and carcass fat in lambs fed PG and lambs fed BD indicates that the inclusion of glucogenic substances did not promote lipogenesis in these lambs fed at or slightly above maintenance.

Adipose Tissue↗
PubMed1989

L-threo-3,4-dihydroxyphenylserine treatment for gait apraxia in parkinsonian patients.

L-threo-3,4-dihydroxyphenylserine (L-DOPS) was administered to six parkinsonian patients for the treatment of gait related akinesia which was refractile to L-DOPA treatment. One responded with marked improvement and one with only mild improvement. Although number of patients who respond markedly to this noradrenaline precursor, L-DOPS, is limited, L-DOPS was felt to be the most effective treatment modality for L-DOPA refractile gait related akinesia and L-DOPA related orthostatic hypotension.

Aged↗
PubMed2000

Negative regulators of hemopoiesis and stroma function in patients with myelodysplastic syndrome.

The mechanism that leads to hemopoietic failure in patients with myelodysplastic syndrome (MDS) is not well understood. There is evidence, however, that regulatory molecules such as tumor necrosis factor (TNF)-alpha, Fas (CD95), and Fas-ligand, which negatively affect hemopoiesis by way of apoptosis are upregulated. Here we analyzed marrow samples from 80 patients with MDS in regard to TNF-alpha and Fas-ligand levels and a possible correlation with various disease parameters and risk factors. TNF-alpha levels were elevated in comparison to samples from normal marrow donors, however, no significant correlation with FAB subtype, cytogenetic risk group or score by the International Prognostic Scoring System (IPSS) was observed. However, there was an inverse correlation between the cytogenetic risk category (low, intermediate, high) and levels of soluble Fas-ligand. The major source of TNF-alpha were mononuclear (non-stromal) cells which appeared to produce TNF-alpha at maximum levels. Limiting dilution analysis of CD34+ precursor cells showed that individually assayed cells, removed from companion cells that presumably provided negative signals such as TNF-alpha or Fas-ligand, were able to generate progressively increasing numbers of colonies. Stromal layers derived from MDS marrow did not have an inhibitory effect. In fact, higher colony numbers were obtained from both normal and MDS marrow derived hemopoietic precursors propagated on irradiated stromal layers from MDS marrow than on stromal layers from normal marrow. These results show that substantial numbers of normal hemopoietic precursors persist in MDS marrow. However, differentiation into mature cells is inhibited by negative signals originating from accessory or abnormal hemopoietic precursors in the non-adherent marrow fraction.

Adolescent↗
PubMed1986

Magnesium deficiency inhibits biosynthesis of blood glutathione and tumor growth in the rat.

Previously we found that blood glutathione (GSH) levels increase in response to tumor growth in the rat and that this increase is not prevented with zinc deficiency. We also found that zinc deficiency which inhibited tumor growth did not prevent this increase in blood GSH. Therefore, the objectives of this study were to determine the effects of another nutritional modification, namely magnesium deficiency, on blood GSH status and on tumor growth. Magnesium was selected because it is an obligatory cofactor in GSH synthesis and in all biosynthetic reactions involving ATP. To this end, magnesium- and zinc-deficient rats with and without tumors were compared to pair-fed control rats with and without tumors. After 32 days of depletion, the rats were killed, and blood samples were analyzed for nonprotein sulfhydryls (SH) and specifically for GSH. The key finding was that in magnesium-deficient rats with or without tumors, blood GSH levels were low and SH levels were normal indicating a decrease in GSH biosynthesis. In contrast, zinc deficiency affected SH and GSH in parallel. Thus, these two deficiencies must act by different mechanisms. The zinc data verified our earlier results obtained with a different tumor type and rat strain, for blood GSH levels increased in tumor-bearing rats fed control diets, and zinc deficiency did not prevent this increase. Depletion of magnesium or zinc was equally effective in inhibiting tumor growth. These results provide in vivo evidence of a magnesium requirement for GSH biosynthesis in rat erythrocytes. Further, the results suggest that magnesium deficiency may inhibit tumor growth by limiting GSH synthesis from SH precursors.

Animals↗
PubMed2001

Depletion of CD4+ T cells precipitates immunopathology in immunodeficient mice infected with a noncytocidal virus.

IFN-gamma-deficient (IFN-gamma(-/-)) mice inoculated with intermediate doses of a slowly replicating strain of lymphocytic choriomeningitis virus become chronically infected. In such mice a hypercompensated CTL response is observed that partially controls virus replication. Here we have investigated whether CD4(+) Th cells are required to establish and maintain this new equilibrium. The absence of IFN-gamma does not impair the generation of IL-2-producing CD4(+) cells, and depletion of these cells precipitates severe CD8(+) T cell-mediated immunopathology in IFN-gamma(-/-) mice, indicating an important role of CD4(+) T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-gamma(-/-) mice following CD4(+) cell depletion. Initially the antiviral CTL response did not require CD4(+) cells, but with time an impaired reactivity toward especially the glycoprotein 33--41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33--41 and nucleoprotein 396--404) CD8(+) T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4(+)-depleted mice, especially in mice also deficient in IFN-gamma. Thus, our findings disclose that T cell help is required to sustain the expanded CTL precursor pool required in IFN-gamma(-/-) mice. This interpretation is supported by mathematical modeling that predicts an increased requirement for help in IFN-gamma(-/-) hosts similar to what is found with fast replicating virus strains in normal hosts. Thus, the functional integrity of CD8(+) effector T cells is one important factor influencing the requirement for T cell help during viral infection.

Acute Disease↗
PubMed1991

[Role of free radicals in bone resorption].

In order to examine the role of the oxygen-derived free radicals in bone metabolism, the mouse calvaria organ culture system and the mouse bone marrow culture system were used. The activity of bone resorption stimulated by 1 alpha, 25(OH)2 vitamin D3 was inhibited by the addition of catalase, an eliminating agent of H2O2. Catalase also significantly suppressed the osteoclast-like cell formation induced by 1 alpha, 25(OH)2 vitamin D3 in a dose-dependent manner, whereas the agent had no effect on the mitogenic activity of the bone marrow-derived stromal cells. This suppression was reversed by adding H2O2 to the system. The effect of catalase was limited to the early stage precursor of the osteoclast-like cells. Moreover, when superoxide dismutase (SOD), which activates the production of H2O2, was added to the bone marrow culture, the number of osteoclast-like cells increased significantly in a dose-dependent manner. Finally, the source of the oxygen-derived free radicals was examined by measuring the fluorescence of 2.7-dichlorofluorescein (DCF), the obtained result indicated that the osteoclast-like cells could produce almost the equivalent amount of H2O2 as could the macrophages. These results strongly suggest that the oxygen-derived free radicals, particularly H2O2, may regulate bone resorption, promoting the differentiation of osteoclast precursor cells to osteoclasts.

Animals↗
PubMedDate not supplied

Analysis of pesticide residues in fruits, vegetables, and milk by gas chromatography/tandem mass spectrometry.

A method for detection, quantitation, and confirmation of more than 100 pesticides by gas chromatography (GC) with ion trap mass spectrometry (MS/MS) has been developed. The sensitivity of this method for many analytes is equal to or lower than those of selective GC detectors such as flame photometric detectors and electrolytic conductivity detectors. Using MS/MS, very low detection limits and good confirmation (1 precursor ion and 2 or more product ions) are achieved simultaneously. The entire list of pesticides is screened with 2 injections per sample. Samples are introduced onto the column by a temperature-programmed cold injection to maximize response. Each pesticide is run with its own unique set of parameters, which fragment the compound, retaining only the precursor ion. This ion is then refragmented to create a product spectrum. The selectivity of MS/MS gives a very clean spectrum, making compound identification and confirmation clear, even with a relatively dirty food matrix. If care is taken to maintain the injection port and guard column, this method can reliably identify and confirm more than 100 pesticides at the low parts-per-billion range.

Animals↗
PubMed1999

Rapid determination of Epstein-Barr virus-specific CD8(+) T-cell frequencies by flow cytometry.

We have developed an efficient and rapid method for detection of Epstein-Barr virus (EBV)-specific CD8(+) T-cell frequencies both in freshly isolated peripheral blood mononuclear cells (PBMCs) and in vitro established cytotoxic T lymphocyte (CTL) lines. Responder cells are thereby stimulated with an autologous lymphoblastoid cell line for 5 hours and intracellular accumulation of interferon gamma (IFNgamma) is detected by multiparameter flow cytometric analysis. EBV-specific CD8(+) T-cell frequencies ranged between 0.63% and 1. 29% in PBMCs of 5 healthy long-term EBV carriers. Using EBV-specific T-cell lines, it was shown that flow cytometric analysis is more sensitive than limiting dilution analysis for CTL precursors and enzyme-linked immunospot assay detecting IFNgamma-producing T cells. The class I restriction of IFNgamma production was confirmed using an anti-class I monoclonal antibody (MoAb). Information on other cytokine production of EBV-specific CTLs could be obtained using combinations of anti-cytokine MoAbs. The sensitive and rapid nature of the flow cytometric assay for EBV-specific CD8(+) T-cell frequency has significant advantages for evaluation of EBV-specific CD8(+) T-cell responses in PBMCs of patients with EBV-related diseases.

Antigens, Viral↗