Isolation and characterization of a B lymphocyte mutant with altered signal transduction through its antigen receptor.

A receptor surface Ig (sIg) signaling variant of WEHI-231 was constructed to investigate components and linkages between various signaling events associated with signal transduction through sIg. Unlike the wildtype, crosslinking of sIgM on VS2.12-cl.2 did not result in downregulation of proliferation. Similarly, receptor crosslinking was uncoupled from inositol phospholipid (PI) hydrolysis and upregulation of c-fos expression in the variant. The signaling defect in VS2.12-cl.2 appears to be proximal to phospholipase C activation as direct G protein activation by A1F4- triggers PI hydrolysis and bypassing PI hydrolysis using phorbol diester stimulation of protein kinase C restores the inhibitable phenotype and the ability to upregulate c-fos. Even more interesting, sIg-linked Ca2+ responses by VS2.12-cl.2 are equivalent to these observed in the wildtype WEHI-231. These latter results suggest that contrary to current thought, sIg-generated signals may not be coupled to Ca2+ fluxes entirely via inositol phospholipid hydrolysis. Thus, VS2.12-cl.2 is a new and powerful tool with which to analyze signaling through sIg at the molecular level.

Signalling through sIgA on a novel murine B lymphoma.

A novel murine B lymphoma expressing membrane-associated IgA was isolated and used to compare mechanisms of signal transduction by sIgM and sIgA. Like other isotypes so far studied, crosslinking of sIgA by anti-immunoglobulin antibodies stimulates hydrolysis of inositol phospholipids and causes elevation of intracellular free calcium. Furthermore, signals generated through sIgA are coupled to elevation of c-fos proto-oncogene expression. Coupling appears to be through the protein kinase C rather than through the Ca2+ component of sIg signalling as phorbol diester, but the Ca2+ ionophore cannot mediate this effect. Thus these results, coupled with those from earlier studies, show that early signal transduction through surface immunoglobulin appears to be similar regardless of the particular isotype involved in binding ligand.

Spectra of intracellular Fura-2.

In the theory of measurement of calcium ion activity by determination of Fura-2 fluorescence at two excitation wavelengths, the accuracy of the result depends upon the accuracy both of the sample measurements and of the calibration measurements which are made on calcium-bound and free dye. Two factors underlie adequate calibration and accuracy. The first is the elimination of systematic error due to spectral shifts arising from the intracellular environment felt by the dye. To this end, detailed comparisons between complete spectra of both calcium-bound and calcium-free Fura-2 can be used to help separate spectral effects due to light absorption by cellular constituents versus polarity and viscosity of the intracellular milieu. The second major factor which determines accuracy is the experimental uncertainty (in both sample and calibration measurements). For samples in which the ratio of bound to free dye is large, the uncertainty in the ratio is also large, even when it is expressed as a percentage of the ratio itself. The errors in calibration measurements impact on the accuracy of the method primarily through the measurements made at wavelengths which are off the spectral peaks of the bound or free dye, since these are the least accurate. In order to obtain a guide to the choice of wavelengths and estimation of the reliability of results, a mathematical expression is derived for the dependence of the accuracy of the method on the accuracy of both sample and calibration measurements.

Quantitation of lymphocyte intracellular free calcium signals using indo-1.

The calcium-responsive fluorescent dye indo-1 has been used in lymphocyte suspensions to measure changes in internal free calcium concentration, [Ca2+]i, in response to crosslinking of cell surface immunoglobulin. The quantitation of [Ca2+]i requires that indo-am ester used to load the cells be completely hydrolyzed to the indo-1 form inside the cells. This is shown to be greatly facilitated in the lymphocyte by the detergent Pluronic F-127. The quantitation of [Ca2+]i transients also requires an estimate of the fraction of the cells which contribute to the observed changes. The use of excessive amounts of intracellular dye can buffer [Ca2+]i transients and this effect has been used to estimate the size of the pool of calcium which is available for release when the B cell is stimulated by anti-immunoglobulin.

Magnetic labeling and cell sorting.

Magnetic labeling by a magnetite-antibody conjugate has been combined with magnetic filtration (high gradient magnetic separation) to effect a rapid and efficient separation of a selected cell population from a suspension of single cells. Samples of more than 10(8) cells could be fractionated in about 5 min with complete recovery. The system has been applied to a model system using red blood cells (sheep or chicken) and commercial antibodies against species-determined cell surface antigens. Enrichments of labeled cells by factors of up to 37-fold were observed. The approach was relatively insensitive to details in the experimental protocol and to the number of unlabeled cells which were in the sample. Thus, the method was easy to use and can readily be scaled up to handle large samples containing 10(8) labeled cells in a total of 10(11) or more. It should be useful as a pre-enrichment scheme for suspensions in which cells of interest are rare and, consequently, very inefficiently sorted by fluorescence-activated instruments.

Magnetic enrichment of antibody-secreting cells.

AMino acid polymer antigens have been covalently coupled to sheep erythrocytes (SRBC) and these have been used in the high gradient magnetic separation (HGMS) technique to enrich murine lymph node cells which had specific receptors for antigen. The method was rapid and provided excellent cell recovery including the fraction retained on the HGMS column. The system could be operated to enrich preformed rosettes, or as an affinity column when preloaded with antigen-coupled SRBC. A 10-fold enrichment of plaque-forming cells was observed in either mode. The HGMS system employed a conventional electromagnet and the fractionation procedure required about 30 min for a sample of up to 10(8) cells.

Effects of 42 degrees C hyperthermia on intracellular pH in ovarian carcinoma cells during acute or chronic exposure to low extracellular pH.

PURPOSE: To determine whether intracellular pH (pHi) is affected during hyperthermia in substrate-attached cells and whether acute extracellular acidification potentiates the cytotoxicity of hyperthermia via an effect on pHi. METHODS AND MATERIALS: The pHi was determined in cells attached to extracellular matrix proteins loaded with the fluorescent indicator dye BCECF at 37 degrees C and during 42 degrees C hyperthermia at an extracellular pH (pHe) of 6.7 or 7.3 in cells. Effects on pHi during hyperthermia are compared to effects on clonogenic survival after hyperthermia at pHe 7.3 and 6.7 of cells grown at pHe 7.3, or of cells grown and monitored at pHe 6.7. RESULTS: The results show that pHi values are affected by substrate attachments. Cells attached to extracellular matrix proteins had better signal stability, low dye leakage and evidence of homeostatic regulation of pHi during heating. The net decrease in pHi in cells grown and assayed at pHe = 7.3 during 42 degrees C hyperthermia was 0.28 units and the decrease in low pH adapted cells heated at pHe = 6.7 was 0.14 units. Acute acidification from pHe = 7.3 to pHe = 6.7 at 37 degrees C caused an initial reduction of 0.5-0.8 unit in pHi, but a partial recovery followed during the next 60-90 min. Concurrent 42 degrees C hyperthermia caused the same initial reduction in pHi in acutely acidified cells, but inhibited the partial recovery that occurred during the next 60-90 min at 37 degrees C. After 4 h at 37 degrees C, the net change in pHi in acutely acidified cells was 0.30 pH unit, but at 42 degrees C is 0.63 pH units. The net change in pHi correlated inversely with clonogenic survival. CONCLUSIONS: Hyperthermia causes a pHi reduction in cells which was smaller in magnitude by 50% in low pH adapted cells. Hyperthermia inhibited the partial recovery from acute acidification that was observed at 37 degrees C in substrate attached cells, in parallel with a lower subsequent clonogenic survival.

Analysis of porphyrins and enzymes in porphyrin synthesis in Taenia solium cysticercus from man and pig.

Porphyrins and activities of heme biosynthetic enzymes in Taenia solium cysticerci from porcine and human hosts, were examined in order to clarify the possible step where heme synthesis is interrupted. Porphyrins in the vesicular fluid of the parasite were predominantly coproporphyrin, followed by penta-carboxylated porphyrin, which together accounted for 90% of the accumulated porphyrins. Coproporphyrin and penta-carboxylated porphyrin were both type I and III isomers. Small amounts of protoporphyrin and uroporphyrin, and trace amounts of tri-, hexa- and hepta-carboxylated porphyrins were also detected. Fluorescence and phosphorescence spectra and lifetime studies revealed that at least 75% of the porphyrins were bound to metal, probably Zn, while the rest was free. Reverse phase high performance liquid chromatography monitored at an excitation wavelength of 417 nm and at an emission wavelength of 585 nm demonstrated that approximately 90% of these porphyrins were Zn-coproporphyrin. A fluorescence excitation peak at 283 nm with an emission peak at 585 nm and 625 nm indicated that some of the porphyrins were associated with proteins in the vesicular fluid of the parasite. Low levels of delta-aminolevulinic acid dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase activities, and heme concentrations were found in the extract of the parasite walls and scolex, but not in the vesicular fluid. The porphyrin accumulation pattern in this parasite can best be explained by postulating a deficiency of coproporphyrinogen oxidase activity, similar to that in human patients with hereditary coproporphyria. A parasite dissected from a human host was considerably less porphyric than those from pigs, but the pattern of accumulated porphyrins was quite similar in both. In view of their porphyrin contents, T. solium cysticerci could be light sensitive.

Synthesis and quantitative structure-activity relationships of dequalinium analogues as K+ channel blockers: investigation into the role of the substituent at position 4 of the quinoline ring.

Dequalinium (4) is a potent and selective blocker of small conductance Ca2+-activated K+ channels, an important but relatively little studied class. The 4-NH2 group of dequalinium has been shown to contribute significantly to blocking potency. In this study, we have investigated further the role of the 4-NH2 group. Replacement of this group by other substituents (R4) and quantitative structure-activity relationship (QSAR) analysis on the resultant analogues have yielded a correlation between blocking potency and sigma R for R4 for seven of the compounds. The application of calculated electronic indices enabled the extension of the QSAR to compounds for which the appropriate sigma R values are not available, allowing all 13 analogues of this series to be included in the correlations. Analysis using electronic indices obtained from AM1 MO calculations on model compounds revealed that the blocking potency correlates with the partial charge on the ring N atom, ELUMO, and EHOMO. The EHOMO correlation is qualitatively inconsistent as the HOMO is not the same orbital in all compounds. The ELUMO correlation [pEMR = 1.19(+/- 0.21)ELUMO + 5.41(+/- 1.05), n = 13, r = 0.86, s = 0.274] suggests that the higher the ELUMO the more potent is the analogue. This is consistent with simple charge transfer from the channel to the blocker and may refer to other processes which are important for the strength of the drug-K+ channel interaction such as the desolvation of the compounds.

Reliable serology of Taenia solium cysticercosis with antigens from cyst vesicular fluid: ELISA and hemagglutination tests.

New levels of reproducibility and sensitivity have been achieved in the detection of anticysticercus antibodies in human sera by using cysticercus vesicular fluid as the source of antigens for both ELISA and hemagglutination assays. Reproducibility both between tests on a serum and between similar sera was significantly improved over typical results using antigens from whole parasite extracts. Sera collected from uninfected individuals in endemic areas gave somewhat elevated values over those collected in nonendemic areas. This necessitated the use of a higher threshold in endemic areas to avoid false positives. With the threshold appropriate for a nonendemic area, both ELISA and hemagglutination were sensitive enough to detect infection in 95% of cases. With the threshold value for sera from an endemic area, these sensitivities were reduced to 80%-90%. A prominent 103-Kd protein of vesicular fluid, not related to antigen B, elicited the strongest antibody response in neurocystercotic patients.

Hypotensive mechanisms of amifostine.

Amifostine, a chemo- and radioprotective agent developed as adjunctive therapy for malignancies, induces hypotension after approximately 20% of patient administrations. This study examines the molecular mechanisms underlying hypotension induced by amifostine. Amifostine and its metabolite, WR-1065, induced dose-dependent hypotension in anesthetized rats that was not blocked by N(G)-methyl L arginine (L-NAME), an NO synthase inhibitor. WR-1065 but not amifostine induced concentration-dependent relaxation of isolated rat aortic rings in an endothelium-independent fashion. Relaxation was not associated with increases in cGMP or cAMP and could not be blocked by L-NAME or indomethacin. Similarly, neither amifostine or WR-1065 activated adenylyl, particulate guanylyl, or soluble guanylyl cyclases. WR-1065 relaxed rat aortic rings precontracted with norepinepherine, suggesting alpha-adrenergic blocking activity. However, neither amifostine nor WR-1065 altered the ability of prazosin or phentolamine to bind to alpha-adrenergic receptors. Further, WR-1065 had no effect on receptor-mediated increases in intracellular calcium in BAL 17 murine B lymphocytes in vitro. Thus, hypotension after administration of amifostine is mediated by WR-1065 and appears to result from direct relaxation of vascular smooth muscle. Smooth muscle relaxation induced by WR-1065 is not related to production of nitric oxide, prostaglandins, or cyclic nucleotides; alpha-adrenergic receptor antagonism; or interference with receptor-dependent increases in intracellular calcium. Administration of ephedrine, an efficacious adrenergic agonist, attenuated hypotension induced by amifostine in anesthetized rats and may be useful in alleviating hypotension associated with amifostine administration in patients.

Immunospecific NMR contrast agents.

Most NMR contrast agents suggested to date have been paramagnetic. These agents, which include the transition and lanthanide metal ions as well as stable organic free radicals, do not provide effective contrast at concentrations much below 1 mM. However, the use of macromolecular ferromagnetic and superparamagnetic particles provides, for the first time, an NMR relaxation agent that is effective at subnanomolar concentrations. Two different sized superparamagnetic particles have been coupled to monoclonal antibodies with high affinity for a neuroblastoma-specific cell surface antigen. The specific binding of these particles, both in vivo and in vitro is demonstrated and the consequences for immunospecific NMR contrast are discussed.

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) partially inhibits rapid intracellular free calcium transients triggered by anti-immunoglobulin in murine lymphocytes.

The highly fluorescent probes Indo-1 and Fura-2 were employed to detect intracellular calcium responses in murine splenic lymphocytes following cross-linking of cell surface Ig. Inhibition by phorbol ester (12-O-tetradecanoylphorbol 13-acetate) was rapid and showed a strong preference for the very transient phase of the response which has been identified as a mobilization of intracellular calcium. 12-O-Tetradecanoylphorbol-13-acetate had significantly less effect on the longer lasting increase in intracellular free calcium which involved an influx of extracellular calcium. Whole spectra were used as a check on transients, which were monitored at a single wavelength, in order to eliminate changes that were not calcium-dependent. It was found that such changes could arise from the association of Indo-1, or its acetoxymethyl ester, with phospholipid bilayers since this affected their fluorescence spectra. In addition, the loading of resting cells with dye esters was shown to be greatly enhanced by the inclusion of a small amount of the detergent Pluronic F-127 in the incubation medium. A spectral analysis of labeled cells showed that the extent of hydrolysis of intracellular dye was improved as well as the rate of uptake by cells.

Magnetic sorting of leukocytes.

Magnetic filtration of surface-labeled cells has been applied to the fractionation of leukocytes in a model system, using a colloidal magnetite reagent to label mouse spleen cells. This reagent was completely free from problems of aggregation or settling. Since the individual submicron particles were invisible under the microscope, cells were not visibly altered by labeling. Viability also was unaffected by either labeling or magnetic filtration. Using a 10-kG magnet and a 5-ml filter column, 50 million cells were fractionated in less than 10 min, with 99% removal of labeled T lymphocytes. The efficiency of the magnetic method is limited at present by the fact that cells that do not have the surface target antigen of interest, and so are not antibody coated, may adsorb a small amount of label nonspecifically. These then have a nonzero chance of being captured in the filter along with the labeled cells.

Simultaneous measurement of two cations with the fluorescent dye indo-1.

The fluorescent chelator Indo-1 can simultaneously provide information about two ions, such as Ca2+ and Ba2+, if spectra after binding can be distinguished from each other and from the ion-free dye. In the case of extracellular Ca2+ and Ba2+, the three spectra were found to be unique and independent, in the sense that no one of them could be approximated by any combination of the other two. Upon binding Ba2+, Indo-1 experienced a peak shift from 470 to 455 nm, whereas the peak for Ca(2+)-bound Indo-1 occurred at 405 nm. The computer could distinguish a contribution from Ba2-bound Indo-1 just as well when it was in a mixture containing contributions from free dye and Ca(2+)-bound Indo-1. The program used all the spectral data in a rapid noniterative calculation. It found the unique combination of Ca(2+)-bound, Ba(2+)-bound, and metal-free Indo-1 spectra which best matched the data. The spectral weighting factors which formed the best combination spectrum then corresponded to the relative concentrations of the dye in the three states. From these and the dissociation constants for Ca2+ and Ba2+, it was possible to calculate both [Ca2+] and [Ba2+] from a single data spectrum. The method was also applied to Mn2+, a metal which does not generate a unique spectrum, but which quenches Indo-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein.

The dyes carboxy-SNARF-1 and BCECF are fluorescent probes of intracellular pH that exhibit changes in spectral shape upon proton binding which allow one to use measurements of fluorescence at two or more wavelengths in order to measure pH without artifacts associated with variability in dye loading, etc. In evaluating these dyes for this study, whole spectra, rather than measurements at two wavelengths, were analyzed. For BCECF, the effects of the intracellular milieu were minimal: both the pH-sensitive excitation spectrum and the pKa agreed closely with values found in extracellular solution. In contrast, both the spectra and the pKa for the emission spectrum-shifting carboxy-SNARF-1 showed significant differences between intracellular and extracellular dye. As a result, extremely misleading values for intracellular pH will be obtained if one attempts to use extracellular dye to calibrate intracellular carboxy-SNARF-1 measurements. Multiple origins were found for the discrepancy: (i) the intracellular dye was found to be significantly quenched, with the deprotonated form being more strongly quenched than the protonated form; and (ii) the pKa for the equilibrium with intracellular hydrogen ions was shifted by +0.2 pH units. These effects were readily reversed by disruption of the cell, but were not due to sequestering of dye in an acidic cell compartment.

Magnetic cell sorting using colloidal protein-magnetite.

Magnetic filtration of labelled cells as a way of separating leucocyte subpopulations was tested with a very simple and easy filtration device, using colloidal magnetite as the labelling reagent. In order to quantitate cell enrichment, a double label (both fluorescent and magnetic) was used, under conditions which labelled less than 10% of the cells in the initial sample. Up to 20 million cells were simply passed through a small magnetic filter with a hand-held syringe. Depletion of labelled cells in the suspension that passed through was threefold, and enrichment of labelled cells in the wash of the filter after its removal from the magnet was approximately fivefold. Factors which limited the quality of separation are discussed. Other, more preliminary, experiments found enrichments of 15- to 30-fold with the same colloidal magnetite and hand-held apparatus when the cell labelling system was more selective.

Dependence of proton generation on aerobic or anaerobic metabolism and implications for tumour pH.

Since low intracellular pH potentiates the hyperthermia response, the number of protons generated within the cell is calculated for both aerobic and anaerobic metabolism. The calculations incorporate the most recent estimates for the P: O ratio in mitochondrial oxidative phosphorylation, and include the hydrolysis of ATP as well as its synthesis. Anaerobic glycolysis of glucose to lactate generates two proton equivalents per mol of glucose, due to the complete dissociation of lactic acid. This corresponds to 1.00 proton per ATP, and is independent of pH. For aerobic metabolism of glucose to CO2 and water, protons are generated in the cell by dissociation of carbonic acid, and the exact number depends upon cytosolic pH. In the physiological pH range, one finds 4.1-5.9 mol of hydrogen ion per mol of glucose, which corresponds to 0.14-0.20 per ATP. Thus, anaerobic metabolism generates 5-7x as much acid within the cell as aerobic metabolism if the two are operating at the same rate in terms of ATP generation per unit time.